Farnesyl dibenzodiazepinone and processes for its production

ABSTRACT

This invention relates to a novel farnesylated dibenzodiazepinone, named ECO-04601, its pharmaceutically acceptable salts and derivatives, and to methods for obtaining such compounds. One method of obtaining the ECO-04601 compound is by cultivation of a novel strain of  Micromonospora  sp., 046-ECO11; another method involves expression of biosynthetic pathway genes in transformed host cells. The present invention further relates to  Micromonospora  sp. strain 046-ECO11, to the use of and its pharmaceutically acceptable salts and derivatives as pharmaceuticals, in particular to their use as inhibitors of cancer cell growth, bacterial cell growth, mammalian lipoxygenase, and to pharmaceutical compositions comprising ECO-04601 or a pharmaceutically acceptable salt or derivative thereof. Finally, the invention relates to novel polynucleotide sequences and their encoded proteins, which are involved in the biosynthesis of ECO-04601.

RELATED APPLICATIONS

This Application is a divisional of U.S. Utility application Ser. No.11/511,586, filed Aug. 28, 2006, which is a divisional of U.S. Utilityapplication Ser. No. 10/762,107, filed Jan. 21, 2004, now issued as U.S.Pat. No. 7,101,872, and which claims priority to each of U.S.Provisional Application 60/441,126, filed Jan. 21, 2003; U.S.Provisional Application 60/492,997, filed Aug. 7, 2003; and U.S.Provisional Application 60/518,286, filed Nov. 10, 2003. The entireteachings of each of the above-noted applications are incorporatedherein by reference.

FIELD OF THE INVENTION

This invention relates to a novel farnesylated dibenzodiazepinone, namedECO-04601, its pharmaceutically acceptable salts and derivatives, and tomethods for obtaining the compound. One method of obtaining the compoundis by cultivation of a novel strain of Micromonospora sp., i.e.,046-ECO11 or [S01]046; another method involves expression ofbiosynthetic pathway genes in transformed host cells. The presentinvention further relates to Micromonospora sp. strains 046-ECO11 and[S01]046, to the use of ECO-04601 and its pharmaceutically acceptablesalts and derivatives as pharmaceuticals, in particular to their use asinhibitors of cancer cell growth, bacterial cell growth, mammalianlipoxygenase, and for treating acute and chronic inflammation, and topharmaceutical compositions comprising ECO-04601 or a pharmaceuticallyacceptable salt or derivative thereof. Finally, the invention relates tonovel polynucleotide sequences and their encoded proteins, which areinvolved in the biosynthesis of ECO-04601.

BACKGROUND OF THE INVENTION

The euactinomycetes are a subset of a large and complex group ofGram-positive bacteria known as actinomycetes. Over the past few decadesthese organisms, which are abundant in soil, have generated significantcommercial and scientific interest as a result of the large number oftherapeutically useful compounds, particularly antibiotics, produced assecondary metabolites. The intensive search for strains able to producenew antibiotics has led to the identification of hundreds of newspecies.

Many of the euactinomycetes, particularly Streptomyces and the closelyrelated Saccharopolyspora genera, have been extensively studied. Both ofthese genera produce a notable diversity of biologically activemetabolites. Because of the commercial significance of these compounds,much is known about the genetics and physiology of these organisms.

Another representative genus of euactinomycetes, Micromonospora, hasalso generated commercial interest. For example, U.S. Pat. No. 5,541,181(Ohkuma et al.) discloses a dibenzodiazepinone compound, specifically5-farnesyl-4,7,9-trihydroxy-dibenzodiazepin-11-one (named “BU-4664L”),produced by a known euactinomycetes strain, Micromonospora sp. M990-6(ATCC 55378). The Ohkurma et al. patent reports that BU-4664L and itschemically synthesized di- and tri-alkoxy and acyloxy derivativespossess anti-inflammatory and anti-tumor cell activities.

Although many biologically active compounds have been identified frombacteria, there remains the need to obtain novel naturally occurringcompounds with enhanced properties. Current methods of obtaining suchcompounds include screening of natural isolates and chemicalmodification of existing compounds, both of which are costly and timeconsuming. Current screening methods are based on general biologicalproperties of the compound, which require prior knowledge of thestructure of the molecules. Methods for chemically modifying knownactive compounds exist, but still suffer from practical limitations asto the type of compounds obtainable.

Thus, there exists a considerable need to obtain pharmaceutically activecompounds in a cost-effective manner and with high yield. The presentinvention solves these problems by providing a novel strain ofMicromonospora capable of producing a potent new therapeutic compound,as well as reagents (e.g., polynucleotides, vectors comprising thepolynucleotides and host cells comprising the vectors) and methods togenerate novel compounds by de novo biosynthesis rather than by chemicalsynthesis.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a compound of the formula

(Formula II) or a pharmaceutically acceptable salt thereof.

In another aspect, the invention relates to a pharmaceutical compositioncomprising a compound of the formula

or a pharmaceutically acceptable salt thereof, together with apharmaceutically acceptable carrier.

In a further aspect, the invention relates to a class of compoundsrepresented by Formula I:

wherein,

-   -   W¹, W² and W³ is each independently selected from

or

the chain from the tricycle may terminate at W³, W² or W¹ with W³, W² orW¹ respectively being either —CH═O or —CH₂OH;

A is selected from —NH—, —NCH₂R¹, —NC(O)R¹;

R¹ is selected from C1-6 alkyl, C2-6 alkene, aryl or heteroaryl;

R², R³, and R⁴ is each independently selected from H, R⁵, —C(O)R⁶

R⁵ is each independently selected from C₁₋₆ alkyl, C₂₋₇ alkalene, arylor heteroaryl;

R⁶ is each independently selected from H, C₁₋₆ alkyl, C₂₋₇ alkalene,aryl or heteroaryl; or a pharmaceutically acceptable salt thereof.

In one embodiment, A is NH.

In another embodiment, A is —NCH₂R¹.

In another embodiment, A is —NC(O)R¹.

In another embodiment, R² is H.

In another embodiment, R³ is H.

In another embodiment, R⁴ is H.

In another embodiment, R², R³ and R⁴ are each H.

In another embodiment, R², R³ and R⁴ are each H, and W¹ is —CH═CH—.

In another embodiment, R², R³ and R⁴ are each H, and W² is —CH═CH—.

In another embodiment, R², R³ and R⁴ are each H, and W³ is —CH═CH—.

In another embodiment, A is NH and R², R³ and R⁴ are each H.

In another embodiment, A is NH, each of W¹, W², and W³ is —CH═CH—.

The invention further encompasses a compound selected from the groupconsisting of:

In one embodiment, the invention relates to compositions of thecompounds of Formula I together with a pharmaceutically acceptablecarrier.

The invention further encompasses a farnesyl dibenzodiazepinone obtainedby a method comprising: a) cultivating Micromonospora sp. strain[S01]046, wherein the cultivation is performed under aerobic conditionsin a nutrient medium comprising at least one source of carbon atoms andat least one source of nitrogen atoms; and b) isolating a farnesyldibenzodiazepinone from the bacteria cultivated in step (a). In oneembodiment the farnesyl dibenzodiazapinone is the compound of FormulaII.

In one embodiment, the farnesyl dibenzodiazepinone generates NMR spectraessentially as shown in FIGS. 3, 4, 5, 6 and 7. In another embodiment,the farnesyl dibenzodiazepinone generates an ¹H NMR spectrum of FIG. 3.

The invention further encompasses a process for making a farnesyldibenzodiazapinone compound, comprising cultivation of Micromonosporasp. strain 046-ECO11, in a nutrient medium comprising at least onesource of carbon atoms and at least one source of nitrogen atoms, andisolation and purification of the compound.

The invention further encompasses a process for making a farnesyldibenzodiazepinone compound comprising cultivation of Micromonospora sp.strain [S01]046 in a nutrient medium comprising at least one source ofcarbon atoms and at least one source of nitrogen atoms, and isolationand purification of the compound.

In one embodiment, the cultivation occurs under aerobic conditions.

In another embodiment, the carbon atom and nitrogen atom sources arechosen from the components shown in Table 16.

In another embodiment, the cultivation is carried out at a temperatureranging from 18° C. to 40° C. In a further embodiment, the temperaturerange is 18° C. to 29° C.

In another embodiment, the cultivation is carried out at a pH rangingfrom 6 to 9.

The invention further encompasses the Micromonospora sp. having IDACAccession No. 231203-01.

The invention further encompasses a method of inhibiting the growth of acancer cell, the method comprising contacting the cancer cell with acompound of Formula I, such that growth of the cancer cell is inhibited.

In one embodiment, the compound is ECO-04601.

The invention further encompasses a method of inhibiting the growth of acancer cell in a mammal, the method comprising administering a compoundof Formula I to a mammal comprising a cancer cell, such that growth ofthe cancer cell is inhibited in the mammal.

In one embodiment, the compound is ECO-04601.

The invention further encompasses a method of treating a pre-cancerousor cancerous condition in a mammal, comprising the step of administeringto the mammal a therapeutically effective amount of a compound ofFormula I, such that a pre-cancerous or cancerous condition is treated.

In one embodiment, the compound is ECO-04601.

The invention further encompasses a method of treating a bacterialinfection in a mammal, comprising administering a therapeuticallyeffective amount of a compound of Formula I to a mammal having abacterial infection, such that the bacterial infection is treated.

In one embodiment, the compound is ECO-04601.

The invention further encompasses a method of reducing inflammation in amammal, comprising administering to a mammal having inflammation atherapeutically effective amount of a compound of Formula I, such thatthe inflammation is reduced.

In one embodiment, the compound is ECO-04601.

The invention further encompasses an isolated polynucleotide comprisingone or more of SEQ ID NOs. 1, 64 and 73, wherein the polynucleotideencodes a polypeptide that participates in a biosynthetic pathway for afarnesyl dibenzodiazepinone.

The invention further encompasses an isolated polynucleotide comprisingSEQ ID NOs. 1, 64 and 73, wherein the polynucleotide encodes apolypeptide that participates in a biosynthetic pathway for a farnesyldibenzodiazepinone.

The invention further encompasses an isolated polynucleotide thatencodes a polypeptide selected from the group consisting of SEQ ID NOs.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78,80, 82, 84, 86 and 88.

In one embodiment, the isolated polynucleotide comprising SEQ ID No. 1encodes a polypeptide selected from the group consisting of SEQ ID Nos.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 56, 58, 60 and 62.

In another embodiment, the isolated polynucleotide comprising SEQ ID No.64 encodes a polypeptide selected from the group consisting of SEQ IDNOS: 65, 67, 69 and 71.

In another embodiment, the isolated polynucleotide comprising SEQ ID No.73, encodes a polypeptide selected from the group consisting of SEQ IDNOS: 74, 76, 78, 80, 82 84, 86 and 88.

The invention further encompasses an isolated polypeptide of SEQ ID NO.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78,80, 82, 84, 86 or 88.

In one embodiment, the polypeptide participates in a biosyntheticpathway for a farnesyl dibenzodiazepinone.

The invention further encompasses an expression vector comprising one ormore of the polynucleotides described herein.

The invention further encompasses a recombinant prokaryotic organismcomprising one or more such expression vectors.

In one embodiment, the organism is an actinomycete.

In another embodiment, the organism requires the expression vector tosynthesize a farnesyl dibenzodiazepinone. That is, the organism isdeficient in the ability to synthesize a farnesyl dibenzodiazepinonebefore transformation with a polynucleotide as described herein.

The invention further encompasses a method of making a farnesyldibenzodiazepinone de novo in a prokaryote, comprising the steps of: (a)providing a prokaryote that is incapable of synthesizing a farnesyldibenzodiazepinone; (b) transforming the prokaryote with an expressionvector as described herein; and (c) culturing the prokaryote; whereinthe culturing results in the synthesis of a farnesyl dibenzodiazepinonein the prokaryote.

In one embodiment, the prokaryote is an actinomycete.

In another embodiment, the vector expresses a polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78,80, 82, 84, 86 or 88.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the mass of ECO-04601 determined by electrospray massspectrometry to be 462.6.

FIG. 2 shows the absorption spectrum of purified ECO-04601 with a UVmaxat 230 nm and a shoulder at 290 nm.

FIG. 3 shows proton NMR data for the compound dissolved in MeOH-d₄.

FIG. 4 shows multidimensional pulse sequences gDQCOSY.

FIG. 5 shows multidimensional pulse sequences gHSQC.

FIG. 6 shows multidimensional pulse sequences gHMBC.

FIG. 7 shows multidimensional pulse sequences NOESY.

FIG. 8 shows the in vitro anti-inflammatory activity of ECO-04601. Graphshows percent inhibition of 5-lipoxygenase activity plotted against theLog μM concentration of ECO-04601 and NDGA. Graph shows the EC₅₀ ofECO-04601 to be 0.93 μM.

FIG. 9 shows inhibition of tumor growth resulting from administration of10 to 30 mg/kg of ECO-04601 to glioblastoma-bearing mice beginning oneday after tumor cell inoculation.

FIG. 10 shows inhibition of tumor growth resulting from administrationof 20-30 mg/kg of ECO-04601 to glioblastoma-bearing mice beginning tendays after tumor cell inoculation.

FIG. 11 shows micrographs of tumor sections from mice bearingglioblastoma tumors and treated with saline or ECO-04601. The celldensity of tumor treated with ECO-04601 appears decreased and nucleifrom ECO-04601-treated tumor cells are larger and pynotic suggesting acytotoxic effect.

FIG. 12 shows the biosynthetic locus of ECO-04601, isolated fromMicromonospora sp. strain 046-ECO11, including the positions of cosmids046KM and 046KQ.

FIG. 13 shows a schematic diagram of the biosynthetic pathway for theproduction of the farnesyl-diphosphate group of ECO-04601 withbiosynthetic enzymes indicated by their ORF number and familydesignation.

FIG. 14 shows a schematic diagram of the biosynthetic pathway for theproduction of (a) 3-hydroxy-anthranilate-adenylate, and (b)2-amino-6-hydroxy-[1,4]benzoquinone components as specified by ORFspresent in the locus encoding ECO-04601. Biosynthetic enzymes areindicated by their ORF number and family designation.

FIG. 15 shows a schematic diagram of the biosynthetic pathway for theassembly of the ECO-04601 precursors, farnesyl-diphosphate,3-hydroxy-anthranilate-adenylate and2-amino-6-hydroxy-[1,4]benzoquinone. Biosynthetic enzymes are indicatedby their ORF number and family designation.

FIG. 16 shows a sequence listing table indicating the SEQ ID NO. andfunction for each of the open reading frames (ORFs) of the 046Dbiosynthetic locus and the corresponding gene product.

FIG. 17 shows results of the fatty acid analysis of Micromonospora sp.strain 046ECO11 (Accession No. IDAC 070303-01). Analysis was conductedusing gas chromatography on fatty acid methyl esters (FAME).

FIG. 18 illustrates the 16 S ribosomal RNA analysis of Micromonosporasp. strain 046ECO11 (Accession No. IDAC 070303-01). Alignment of 16 Sribosomal RNA sequences demonstrates the phylogenetic relatedness ofMicromonospora sp. strain 046ECO11 (indicated as MID352 ECOPIA#1 con) toMicromonospora chalcea.

FIG. 19 shows the complete ¹H and ¹³C NMR assignments for ECO-04601 whenmeasured in MeOH-d4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel farnesyl dibenzodiazepinone,referred to herein as “ECO-04601,” which was isolated from novel strainsof actinomycetes, Micromonospora sp. strain 046-ECO11 and strain[S01]046. These microorganisms were analysed using gas chromatography asFatty acid methyl esters (FAME) (FIG. 17) 6 S ribosomal RNAdetermination (FIG. 18) and were found to belong to the genus ofMicromonospora. These organisms were deposited on Mar. 7, 2003, and Dec.23, 2003, respectively, with the International Depository Authority ofCanada (IDAC), Bureau of Microbiology, Health Canada, 1015 ArlingtonStreet, Winnipeg, Manitoba, Canada R3E 3R2, under Accession Nos. IDAC070303-01 and IDAC 231203-01, respectively.

The invention further relates to pharmaceutically acceptable salts andderivatives of ECO-04601, and to methods for obtaining such compounds.One method of obtaining the compound is by cultivating Micromonosporasp. strain 046-ECO11, or a mutant or a variant thereof, under suitableMicromonospora culture conditions, preferably using the fermentationprotocol described hereinbelow.

The invention also relates to a method for producing novel polyketidecompounds, namely farnesyl dibenzodiazepinones, by selectively alteringthe genetic information of an organism. The present invention furtherprovides isolated and purified polynucleotides that encode farnesyldibenzodiazepinone domains, i.e., polypeptides from farnesyldibenzodiazepinone-producing microorganisms, fragments thereof, vectorscontaining those polynucleotides, and host cells transformed with thosevectors. These polynucleotides, fragments thereof, and vectorscomprising the polynucleotides can be used as reagents in the abovedescribed method. Portions of the polynucleotide sequences disclosedherein are also useful as primers for the amplification of DNA or asprobes to identify related domains from other farnesyldibenzodiazepinone producing microorganisms.

The present invention also relates to pharmaceutical compositionscomprising ECO-04601 and its pharmaceutically acceptable salts andderivatives. ECO-04601 is useful as a pharmaceutical, in particular foruse as an inhibitor of cancer cell growth, bacterial cell growth, andmammalian lipoxygenase. The invention also relates to novelpolynucleotide sequences and their encoded proteins, which are involvedin the biosynthesis of ECO-04601.

The following detailed description discloses how to make and useECO-04601 and compositions containing this compound to inhibit microbialgrowth and/or specific disease pathways.

Accordingly, certain aspects of the present invention relate topharmaceutical compositions comprising the farnesylateddibenzodiazepinone compounds of the present invention together with apharmaceutically acceptable carrier, methods of using the compositionsto inhibit bacterial growth, and methods of using the pharmaceuticalcompositions to treat diseases, including cancer, and chronic and acuteinflammation.

I. Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below.

As used herein, the term “farnesyl dibenzodiazepinone” refers to a classof dibenzodiazepinone compounds containing a farnesyl moiety. The termincludes, but is not limited to, the exemplified compound of the presentinvention, 10-farnesyl-4,6,8-trihydroxy-dibenzodiazepin-11-one, which isreferred to herein as “ECO-04601.” As used herein, the term “farnesyldibenzodiazepinone” includes compounds of this class that can be used asintermediates in chemical syntheses. As used herein, the term “alkyl”refers to linear or branched hydrocarbon groups. Examples of alkylgroups include, without limitation, methyl, ethyl, n-propyl, isopropyl,n-butyl, pentyl, hexyl, heptyl, cyclopentyl, cyclohexyl,cyclohexymethyl, and the like. Alkyl may optionally be substituted withsubstituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy,carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy,aryloxy, sulfinyl, sulfonyl, oxo, guanidino and formyl.

The term “alkenyl” refers to linear, branched or cyclic hydrocarbongroups containing at least one carbon-carbon double bond. Examples ofalkenyl groups include, without limitation, vinyl, 1-propen-2-yl,1-buten-4-yl, 2-buten-4-yl, 1-penten-5-yl and the like. Alkenyl mayoptionally be substituted with substituents selected from acyl, amino,acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo,hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl,formyl, oxo and guanidino. The double bond portion(s) of the unsaturatedhydrocarbon chain may be either in the cis or trans configuration.

The terms “cycloalkyl” and “cycloalkyl ring” refer to a saturated orpartially unsaturated carbocyclic ring in a single or fused carbocyclicring system having from three to fifteen ring members. Examples ofcycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclohexyl, and cycloheptyl. Cycloalkyl may optionally be substitutedwith substituents selected from acyl, amino, acylamino, acyloxy,carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.

The terms “heterocyclyl” and “heterocyclic” refer to a saturated orpartially unsaturated ring containing one to four hetero atoms or heterogroups selected from O, N, NH, NRx, PO2, S, SO or SO₂ in a single orfused heterocyclic ring system having from three to fifteen ringmembers. Examples of a heterocyclyl or heterocyclic ring include,without limitation, morpholinyl, piperidinyl, and pyrrolidinyl.Heterocyclyl, heterocyclic or heterocyclyl ring may optionally besubstituted with substituents selected from acyl, amino, acylamino,acyloxy, oxo, thiocarbonyl, imino, carboalkoxy, carboxy, carboxyamido,cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl andformyl.

The term “amino acid” refers to any natural amino acid, all naturalamino acids are well known to a person skilled in the art.

The term “halo” refers to a halogen atom, e.g., bromine, chlorine,fluorine and iodine.

The terms “aryl” and “aryl ring” refer to aromatic groups in a single orfused ring system, having from five to fifteen ring members. Examples ofaryl include, without limitation, phenyl, naphthyl, biphenyl, terphenyl.Aryl may optionally be substituted with one or more substituent groupselected from acyl, amino, acylamino, acyloxy, azido, alkylthio,carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.

The terms “heteroaryl” and “heteroaryl ring” refer to aromatic groups ina single or fused ring system, having from five to fifteen ring membersand containing at least one hetero atom such as O, N, S, SO and SO₂.Examples of heteroaryl groups include, without limitation, pyridinyl,thiazolyl, thiadiazoyl, isoquinolinyl, pyrazolyl, oxazolyl, oxadiazoyl,triazolyl, and pyrrolyl groups. Heteroaryl groups may optionally besubstituted with one or more substituent group selected from acyl,amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano,halo, hydroxyl, nitro, thio, thiocarbonyl, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl,sulfonyl, and formyl.

The terms “aralkyl” and “heteroaralkyl” refer to an aryl group or aheteroaryl group, respectively bonded directly through an alkyl group,such as benzyl. Aralkyl and heteroaralkyl may be optionally substitutedas the aryl and heteroaryl groups.

Similarly, the terms “aralkenyl” and “heteroaralkenyl” refer to an arylgroup or a heteroaryl group, respectively bonded directly through analkene group, such as benzyl. Aralkenyl and heteroaralkenyl may beoptionally substituted as the aryl and heteroaryl groups.

The compounds of the present invention can possess one or moreasymmetric carbon atoms and can exist as optical isomers formingmixtures of racemic or non-racemic compounds. The compounds of thepresent invention are useful as single isomers or as a mixture ofstereochemical isomeric forms. Diastereoisomers, i.e., nonsuperimposablestereochemical isomers, can be separated by conventional means such aschromatography, distillation, crystallization or sublimation. Theoptical isomers can be obtained by resolution of the racemic mixturesaccording to conventional processes.

The invention encompasses isolated or purified compounds. An “isolated”or “purified” compound refers to a compound which represents at least10%, 20%, 50%, 80% or 90% of the compound of the present inventionpresent in a mixture, provided that the mixture comprising the compoundof the invention has demonstrable (i.e. statistically significant)biological activity including antibacterial, cytostatic, cytotoxic,antiinflammatory or enzyme inhibitory action when tested in conventionalbiological assays known to a person skilled in the art.

The terms “farnesyl dibenzodiazepinone-producing microorganism” and“producer of farnesyl dibenzodiazepinone,” as used herein, refer to amicroorganism that carries genetic information necessary to produce afarnesyl dibenzodiazepinone compound, whether or not the organismnaturally produces the compound. The terms apply equally to organisms inwhich the genetic information to produce the farnesyl dibenzodiazepinonecompound is found in the organism as it exists in its naturalenvironment, and to organisms in which the genetic information isintroduced by recombinant techniques.

Specific organisms contemplated herein include, without limitation,organisms of the family Micromonosporaceae, of which preferred generainclude Micromonospora, Actinoplanes and Dactylosporangium; the familyStreptomycetaceae, of which preferred genera include Streptomyces andKitasatospora; the family Pseudonocardiaceae, of which preferred generaare Amycolatopsis and Saccharopolyspora; and the familyActinosynnemataceae, of which preferred genera include Saccharothrix andActinosynnema; however the terms are intended to encompass all organismscontaining genetic information necessary to produce a farnesyldibenzodiazepinone compound. A preferred producer of a farnesyldibenzodiazepinone compound includes microbial strain 046-ECO11, adeposit of which was made on Mar. 7, 2003, with the InternationalDepository Authority of Canada (IDAC), Bureau of Microbiology, HealthCanada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E, 3R2,under Accession No. IDAC 070303-01.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as, where applicable,intervening regions (introns) between individual coding segments(exons).

The terms “gene locus, “gene cluster,” and “biosynthetic locus” refer toa group of genes or variants thereof involved in the biosynthesis of afarnesyl benzodiazepinone compound. The biosynthetic locus in strain046-ECO11 that directs the production of ECO-04601 is often referred toherein, in both the written description and Figures, as “046D.” Geneticmodification of gene locus, gene cluster or biosynthetic locus refers toany genetic recombinant techniques known in the art includingmutagenesis, inactivation, or replacement of nucleic acids that can beapplied to generate variants of ECO-04601.

A DNA or nucleotide “coding sequence” or “sequence encoding” aparticular polypeptide or protein, is a DNA sequence which istranscribed and translated into a polypeptide or protein when placedunder the control of an appropriate regulatory sequence.

“Oligonucleotide” refers to a nucleic acid, generally of at least 10,preferably 15 and more preferably at least 20 nucleotides in length,preferably no more than 100 nucleotides in length, that are hybridizableto a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encodinga gene, mRNA, cDNA or other nucleic acid of interest.

A promoter sequence is “operably linked to” a coding sequence recognizedby RNA polymerase which initiates transcription at the promoter andtranscribes the coding sequence into mRNA.

The term “replicon” as used herein means any genetic element, such as aplasmid, cosmid, chromosome or virus, that behaves as an autonomous unitof polynucleotide replication within a cell. A “expression vector” or“vector” is a replicon in which another polynucleotide fragment isattached, such as to bring about the replication and/or expression ofthe attached fragment. “Plasmids” are designated herein by a lower case“p” preceded or followed by capital letters and/or numbers. The startingplasmids disclosed herein are commercially available, publicly availableon an unrestricted basis, or can be constructed from available plasmidsin accordance with published procedures. In addition, equivalentplasmids to those described herein are known in the art and will beapparent to the skilled artisan.

The terms “express” and “expression” means allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell. Anexpression product can be characterized as intracellular, extracellularor secreted.

“Digestion” of DNA refers to enzymatic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinary skilled artisan. For analytical purposes,typically 1 μg of plasmid or DNA fragment is used with about 2 units ofenzyme in about 20 μl of buffer solution. For the purpose of isolatingDNA fragments for plasmid construction, typically 5 to 50 μg of DNA aredigested with 20 to 250 units of enzyme in a larger volume. Appropriatebuffers and substrate amounts for particular enzymes are specified bythe manufacturer. Incubation times of about 1 hour at 37° C. areordinarily used, but may vary in accordance with the supplier'sinstructions. After digestion the gel electrophoresis may be performedto isolate the desired fragment.

The term “isolated” as used herein means that the material is removedfrom its original environment (e.g. the natural environment where thematerial is naturally occurring). For example, a naturally occurringpolynucleotide or polypeptide present in a living animal is notisolated, but the same polynucleotide or polypeptide, which is separatedfrom some or all of the coexisting materials in the natural system, isisolated. Such polynucleotides could be part of a vector and/or suchpolynucleotides or polypeptides could be part of a composition, andstill be isolated in that the vector or composition is not part of thenatural environment.

The term “restriction fragment” as used herein refers to any linear DNAgenerated by the action of one or more restriction enzymes.

The term “transformation” means the introduction of a foreign gene,foreign nucleic acid, DNA or RNA sequence to a host cell, so that thehost cell will express the introduced gene or sequence to produce adesired substance, typically a protein or enzyme coded by the introducedgene or sequence. The introduced gene or sequence may also be called a“cloned” or “foreign” gene or sequence, may include regulatory orcontrol sequences, such as start, stop, promoter, signal, secretion, orother sequences used by a cell's genetic machinery. The gene or sequencemay include nonfunctional sequences or sequences with no known function.A host cell that receives and expresses introduced DNA or RNA has been“transformed” and is a “transformant” or a “clone” or “recombinant”. TheDNA or RNA introduced to a host cell can come from any source, includingcells of the same genus or species as the host cell, or cells of adifferent genus or species.

The terms “recombinant polynucleotide” and “recombinant polypeptide” asused herein mean a polynucleotide or polypeptide which by virtue of itsorigin or manipulation is not associated with all or a portion of thepolynucleotide or polypeptide with which it is associated in natureand/or is linked to a polynucleotide or polypeptide other than that towhich it is linked in nature.

The term “host cell” as used herein, refer to both prokaryotic andeukaryotic cells which are used as recipients of the recombinantpolynucleotides and vectors provided herein. In one embodiment, the hostcell is a prokaryote.

The terms “open reading frame” and “ORF” as used herein refers to aregion of a polynucleotide sequence which encodes a polypeptide; thisregion may represent a portion of a coding sequence or a total codingsequence.

As used herein and as known in the art, the term “identity” is therelationship between two or more polynucleotide sequences, as determinedby comparing the sequences. Identity also means the degree of sequencerelatedness between polynucleotide sequences, as determined by the matchbetween strings of such sequences. Identity can be readily calculated(see, e.g., Computation Molecular Biology, Lesk, A. M., eds., OxfordUniversity Press, New York (1998), and Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York (1993),both of which are incorporated by reference herein). While there exist anumber of methods to measure identity between two polynucleotidesequences, the term is well known to skilled artisans (see, e.g.,Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press(1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J.,eds., M. Stockton Press, New York (1991)). Methods commonly employed todetermine identity between sequences include, for example, thosedisclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math. (1988)48:1073. “Substantially identical,” as used herein, means there is avery high degree of homology (preferably 100% sequence identity) betweensubject polynucleotide sequences. However, polynucleotides havinggreater than 90%, or 95% sequence identity may be used in the presentinvention, and thus sequence variations that might be expected due togenetic mutation, strain polymorphism, or evolutionary divergence can betolerated.

As used herein, the term “treatment” refers to the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disorder, e.g., a disease or condition, asymptom of disease, or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve, or affect the disease, the symptoms of disease, or thepredisposition toward disease.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a farnesyl dibenzodiazepinone anda pharmaceutically acceptable carrier. As used herein,“pharmacologically effective amount,” “therapeutically effective amount”or simply “effective amount” refers to that amount of a farnesyldibenzodiazepinone effective to produce the intended pharmacological,therapeutic or preventive result. For example, if a given clinicaltreatment is considered effective when there is at least a 25% reductionin a measurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 25%reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

The term “pharmaceutically acceptable salt” refers to both acid additionsalts and base addition salts. The nature of the salt is not critical,provided that it is pharmaceutically acceptable. Exemplary acid additionsalts include, without limitation, hydrochloric, hydrobromic,hydroiodic, nitric, carbonic, sulphuric, phosphoric, formic, acetic,citric, tartaric, succinic, oxalic, malic, glutamic, propionic,glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic,toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic,algenic, β-hydroxybutyric, malonic, galactaric, galacturonic acid andthe like. Suitable pharmaceutically acceptable base addition saltsinclude, without limitation, metallic salts made from aluminium,calcium, lithium, magnesium, potassium, sodium and zinc or organic saltsmade from N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, N-methylglucamine, lysine, procaine andthe like. Additional examples of pharmaceutically acceptable salts arelisted in Journal of Pharmaceutical Sciences (1977) 66:2. All of thesesalts may be prepared by conventional means from a farnesyldibenzodiazepinone by treating the compound with the appropriate acid orbase.

II. Farnesylated Dibenzodiazepinone Compounds

In one aspect, the invention relates to a novel farnesyldibenzodiazepinone, referred to herein as “ECO-04601” and having thechemical structure represented by the following formula:

ECO-04601 may be described as a new dibenzodiazepinone having a10-farnesyl substituent located on the nitrogen atom in the 10 positionof the dibenzodiazepine ring (i.e., the amide nitrogen in thediazepinone ring), and three phenolic hydroxy substituents in the 4,6and 8 positions of the dibenzodiazepinone ring. ECO-04601 may becharacterized by any one or more of its physicochemical and spectralproperties given below, such as its mass, UV, and NMR spectroscopicdata. Mass was determined by electrospray mass spectrometry to be 462.6(FIG. 1); UV=230 nm with a shoulder at 290 nm (FIG. 2). NMR data werecollected using MeOH-d4, including proton (FIG. 3), and multidimensionalpulse sequences gDQCOSY (FIG. 4), gHSQC (FIG. 5), gHMBC (FIG. 6), andNOESY (FIG. 7).

In another aspect, the invention relates to a novel class of farnesyldibenzodiazepinone compounds represented by Formula I:

wherein,

-   -   W¹, W² and W³ is each independently selected from

or

the chain from the tricycle may terminate at W³, W² or W¹ with W³, W² orW¹ respectively being either —CH═O or —CH₂OH;

A is selected from —NH—, —NCH₂R¹, —NC(O)R¹;

R¹ is selected from C1-6 alkyl, C2-6 alkene, aryl or heteroaryl;

R², R³, and R⁴ is each independently selected from H, R⁵, —C(O)R⁶

R⁵ is each independently selected from C₁₋₆ alkyl, C₂₋₇ alkalene, arylor heteroaryl;

R⁶ is each independently selected from H, C₁₋₆ alkyl, C₂₋₇ alkalene,aryl or heteroaryl; or a pharmaceutically acceptable salt thereof.

In other embodiments, the invention provides compounds of Formula I,wherein A is selected from the group consisting of NH, NCH2R1, andNC(O)R1; wherein R2 is H; R3 is H; and R4 is H. In another embodiment,R2, R3 and R4 are each H; and all other groups are as previouslydefined. In a further embodiment, R2, R3 and R4 are each H; and W1 is—CH═CH— and all other groups are as previously defined. In a furtherembodiment, R2, R3 and R4 are each H, and W2 is —CH═CH— and all othergroups are as previously defined. In a further embodiment, R2, R3 and R4are each H; and W3 is —CH═CH—; and all other groups are as previouslydefined. In a further embodiment, A is NH; R2, R3 and R4 are each H; andall other groups are as previously defined. In a further embodiment, Ais NH; each of W1, W2, and W3 is —CH ═CH—; and all other groups are aspreviously defined. The invention encompasses all pharmaceuticallyacceptable salts of the foregoing compounds.

The following are exemplary compounds of the invention:

Certain embodiments expressly exclude one or more of the compounds ofFormula I. In one embodiment, the compound of Formula II is excluded.

The compounds of this invention may be formulated into pharmaceuticalcompositions comprised of compounds of Formula I in combination with apharmaceutical acceptable carrier, as discussed in Section V below.

III. Method of Making a Farnesyl Dibenzodiazepinone by Fermentation

In one embodiment, ECO-04601 is obtained by cultivating a novel strainof Micromonospora, namely Micromonospora sp. strain 046-ECO11. Strain046-ECO11 was deposited on Mar. 7, 2003, with the InternationalDepositary Authority of Canada (IDAC), Bureau of Microbiology, HealthCanada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2, underAccession No. 070303-01. The deposit of the strain was made under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for Purposes of Patent Procedure. Thedeposited strains will be irrevocably and without restriction orcondition released to the public upon the issuance of a patent. Thedeposited strains are provided merely as convenience to those skilled inthe art and are not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

It is to be understood that the present invention is not limited to useof the particular strain 046-ECO11. Rather, the present inventioncontemplates the use of other ECO-04601 producing organisms, such asmutants or variants of 046-ECO11 that can be derived from this organismby known means such as X-ray irradiation, ultraviolet irradiation,treatment with nitrogen mustard, phage exposure, antibiotic selectionand the like; or through the use of recombinant genetic engineeringtechniques, as described in Section IV below.

The farnesyl dibenzodiazepinone compounds of the present invention maybe biosynthesized by various microorganisms. Microorganisms that maysynthesize the compounds of the present invention include but are notlimited to bacteria of the order Actinomycetales, also referred to asactinomycetes. Non-limiting examples of members belonging to the generaof Actinomycetes include Nocardia, Geodermatophilus, Actinoplanes,Micromonospora, Nocardioides, Saccharothrix, Amycolatopsis, Kutzneria,Saccharomonospora, Saccharopolyspora, Kitasatospora, Streptomyces,Microbispora, Streptosporangium, and Actinomadura. The taxonomy ofactinomycetes is complex and reference is made to Goodfellow,Suprageneric Classification of Actinomycetes (1989); Bergey's Manual ofSystematic Bacteriology, Vol. 4 (Williams and Wilkins, Baltimore, pp.2322-2339); and to Embley and Stackebrandt, “The molecular phylogeny andsystematics of the actinomycetes,” Annu. Rev. Microbiol. (1994)48:257-289, each of which is hereby incorporated by reference in itsentirety, for genera that may synthesize the compounds of the invention.

Farnesyl dibenzodiazepinone-producing microorganisms are cultivated inculture medium containing known nutritional sources for actinomycetes.Such media having assimilable sources of carbon, nitrogen, plus optionalinorganic salts and other known growth factors at a pH of about 6 toabout 9. Suitable media include, without limitation, the growth mediaprovided in Table 16. Microorganisms are cultivated at incubationtemperatures of about 18° C. to about 40° C. for about 3 to about 40days.

The culture media inoculated with the farnesyldibenzodiazepinone-producing microorganisms may be aerated by incubatingthe inoculated culture media with agitation, for example, shaking on arotary shaker, or a shaking water bath. Aeration may also be achieved bythe injection of air, oxygen or an appropriate gaseous mixture to theinoculated culture media during incubation. Following cultivation, thefarnesyl dibenzodiazepinone compounds can be extracted and isolated fromthe cultivated culture media by techniques known to a skilled person inthe art and/or disclosed herein, including for example centrifugation,chromatography, adsorption, filtration. For example, the cultivatedculture media can be mixed with a suitable organic solvent such asn-butanol, n-butyl acetate or 4-methyl-2-pentanone, the organic layercan be separated for example, by centrifugation followed by the removalof the solvent, by evaporation to dryness or by evaporation to drynessunder vacuum. The resulting residue can optionally be reconstituted withfor example water, ethanol, ethyl acetate, methanol or a mixturethereof, and re-extracted with a suitable organic solvent such ashexane, carbon tetrachloride, methylene chloride or a mixture thereof.Following removal of the solvent, the compounds may be further purifiedby the use of standard techniques, such as chromatography.

The farnesyl dibenzodiapezinones biosynthesized by microorganisms mayoptionally be subjected to random and/or directed chemical modificationsto form compounds that are derivatives or structural analogs. Suchderivatives or structural analogs having similar functional activitiesare within the scope of the present invention. Farnesyldibenzodiapezinone compounds may optionally be modified using methodsknown in the art and described herein.

IV. Method of Making a Farnesyl Dibenzodiazepinone by RecombinantTechnology

In another embodiment, the present invention relates to nucleic acidmolecules that encode proteins useful in the production of farnesylbenzodiazepinones. Specifically, the present invention providesrecombinant DNA vectors and nucleic acid molecules that encode all orpart of the biosynthetic locus in strain 046-ECO11, which directs theproduction of ECO-04601, and is referred to herein as “046D.” Theinvention further includes genetic modification of 046D usingconventional genetic recombinant techniques, such as mutagenesis,inactivation, or replacement of nucleic acids, to produce chemicalvariants of ECO-04601.

The invention thus provides a method for making a farnesylbenzodiazepinone compound using a transformed host cell comprising arecombinant DNA vector that encodes one or more of the polypeptides ofthe present invention, and culturing the host cell under conditions suchthat farnesyl benzodiazepinone is produced. The host cell is aprokaryote. In one embodiment, the host cell is an actinomycete. Inanother embodiment, the host cell is a Streptomyces host cell.

The invention provides recombinant nucleic acids that produce a varietyof farnesyl dibenzodiazepinone compounds that cannot be readilysynthesized by chemical methodology alone. The invention allows directmanipulation of 046D biosynthetic locus via genetic engineering of theenzymes involved in the biosynthesis of a farnesyl benzodiazepinoneaccording to the invention. The 046A biosynthetic locus is described inExample 11.

Recombinant DNA Vectors

Vectors of the invention typically comprise the DNA of a transmissibleagent, into which foreign DNA is inserted. A common way to insert onesegment of DNA into another segment of DNA involves the use of specificenzymes called restriction enzymes that cleave DNA at specific sites(specific groups of nucleotides) called restriction sites. A “cassette”refers to a DNA coding sequence or segment of DNA that codes for anexpression product that can be inserted into a vector at definedrestriction sites. The cassette restriction sites are designed to ensureinsertion of the cassette in the proper reading frame. Generally, anucleic acid molecule that encodes a protein useful in the production ofa farnesyl benzodiazepinone is inserted at one or more restriction sitesof the vector DNA, and then is carried by the vector into a prokaryotee.g. actinomycte, by transformation (see below). A segment or sequenceof DNA having inserted or added DNA, such as an expression vector, canalso be called a “DNA construct”. A common type of vector is a “plasmid”which generally is a self-contained molecule of double-stranded DNA,usually of bacterial origin, that can readily accept additional(foreign) DNA and which can be readily introduced into a suitable hostcell. A plasmid vector often contains coding DNA and promoter DNA andhas one or more restriction sites suitable for inserting foreign DNA.Coding DNA is a DNA sequence that encodes a particular amino acidsequence for a particular protein or enzyme. In one embodiment of theinvention, the coding DNA encodes for polypeptides of SEQ ID NOs. 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79,81, 83, 85, 87 or 89 that are required for the biosynthesis of afarnesyl benzodiazepinone.

Promoter DNA of a recombinant vector is a DNA sequence that initiates,regulates, or otherwise mediates or controls the expression of thecoding DNA. Promoter DNA and coding may be from the same or differentorganisms. Recombinant cloning vectors will often include one or morereplication systems for cloning or expression, one or more markers forselection in the host, e.g. antibiotic resistance, and one or moreexpression cassettes. Vector constructs may be produced usingconventional molecular biology and recombinant DNA techniques within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A LaboratoryManual, Second Edition (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (herein “Sambrook et al., 1989”); DNA Cloning: APractical Approach, Volumes I and II (D. N. Glover ed. 1985); F. M.Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, Inc. (1994).

Examples of promoters that function in actinomycetes, e.g. Streptomyces,are taught in U.S. Pat. Nos. 5,830,695 and 5,466,590. Another example ofa transcription promoter useful in Actinomycetes expression vectors istipA, a promoter inducible by the antibiotic thiostrepton [c.f.Murakami, T., et al., (1989), J. Bacteriol., 171,1459].

Transformation of Actinomycetes

A suitable transformation method for use with an actinomycete comprisesforming the actinomycete culture into spheroplasts using lysozyme. Abuffer solution containing recombinant DNA vectors and polyethyleneglycol is then added, in order to introduce the vector into the hostcells, by using either of the methods of Thompson or Keiser [c. f.Thompson, C. J., et al., (1982), J. Bacteriol., 151, 668-677 or Keiser,T. et al. (2000), “Practical Streptomyces Genetics”, The John InnesFoundation, Norwich], for example. A thiostrepton-resistance gene isfrequently used as a selective marker in the transformation plasmid[c.f. Hopwood, D. A., et al., (1987), “Methods in Enzymology” 153, 116,Academic Press, New York], but the present invention is not limitedthereto. Additional methods for the transformation of actinomycetes aretaught in U.S. Pat. No. 5,393,665.

Assay for Farnesyl Dibenzodiazepinone or Biosynthetic Intermediates

Actinomycetes defective in farnesyl dibenzodiazepinone biosynthesis aretransformed with one or more expression vectors encoding one or moreproteins in the farnesyl benzodiazepinone biosynthetic pathway, thusrestoring farnesyl benzodiazepinone biosynthesis by geneticcomplementation of the specific defect.

The presence or absence of farnesyl dibenzodiazepinone or intermediatesin the biosynthetic pathway (see FIGS. 13, 14 and 15) in a recombinantactinomycete can be determined using methodologies that are well knownto persons of skill in the art. For example, ethyl acetate extracts offermentation media used for the culture of a recombinant actinomyceteare processed as described in Example 2 and fractions containingfarnesyl dibenzodiazepinone or intermediates detected by TLC oncommercial Kieselgel 60F₂₅₄ plates. Farnesyl dibenzodiazepinone andintermediate compounds are visualized by inspection of dried platesunder UV light or by spraying the plates with a spray containingvanillin (0.75%) and concentrated sulfuric acid (1.5%, v/v) in ethanoland subsequently heating the plate. The exact identity of the compoundsseparated by TLC is then determined using gas chromatography-massspectroscopy. Methods of mass spectroscopy are taught in the publishedU.S. Patent Application No. US2003/0052268.

Mutagenesis

The invention allows direct manipulation of 046D biosynthetic locus viagenetic engineering of the enzymes involved in the biosynthesis of afarnesyl benzodiazepinone according to the invention.

A number of methods are known in the art that permit the random as wellas targeted mutation of the DNA sequences of the invention (see forexample, Ausubel et. al. Short Protocols in Molecular Biology (1995) 3rdEd. John Wiley & Sons, Inc.). In addition, there are a number ofcommercially available kits for site-directed mutagenesis, includingboth conventional and PCR-based methods. Examples include the EXSITE™PCR-Based Site-directed Mutagenesis Kit available from Stratagene(Catalog No. 200502) and the QUIKCHANGE™ Site-directed mutagenesis Kitfrom Stratagene (Catalog No. 200518), and the CHAMELEON® double-strandedSite-directed mutagenesis kit, also from Stratagene (Catalog No.200509).

In addition the nucleotides of the invention may be generated byinsertional mutation or truncation (N-terminal, internal or C-terminal)according to methodology known to a person skilled in the art.

Older methods of site-directed mutagenesis known in the art rely onsub-cloning of the sequence to be mutated into a vector, such as an M13bacteriophage vector, that allows the isolation of single-stranded DNAtemplate. In these methods, one anneals a mutagenic primer (i.e., aprimer capable of annealing to the site to be mutated but bearing one ormore mismatched nucleotides at the site to be mutated) to thesingle-stranded template and then polymerizes the complement of thetemplate starting from the 3′ end of the mutagenic primer. The resultingduplexes are then transformed into host bacteria and plaques arescreened for the desired mutation.

More recently, site-directed mutagenesis has employed PCR methodologies,which have the advantage of not requiring a single-stranded template. Inaddition, methods have been developed that do not require sub-cloning.Several issues must be considered when PCR-based site-directedmutagenesis is performed. First, in these methods it is desirable toreduce the number of PCR cycles to prevent expansion of undesiredmutations introduced by the polymerase. Second, a selection must beemployed in order to reduce the number of non-mutated parental moleculespersisting in the reaction. Third, an extended-length PCR method ispreferred in order to allow the use of a single PCR primer set. Andfourth, because of the non-template-dependent terminal extensionactivity of some thermostable polymerases it is often necessary toincorporate an end-polishing step into the procedure prior to blunt-endligation of the PCR-generated mutant product.

The protocol described below accommodates these considerations throughthe following steps. First, the template concentration used isapproximately 1000-fold higher than that used in conventional PCRreactions, allowing a reduction in the number of cycles from 25-30 downto 5-10 without dramatically reducing product yield. Second, therestriction endonuclease Dpn I (recognition target sequence: 5-Gm6ATC-3,where the A residue is methylated) is used to select against parentalDNA, since most common strains of E. coli Dam methylate their DNA at thesequence 5-GATC-3. Third, Taq Extender is used in the PCR mix in orderto increase the proportion of long (i.e., full plasmid length) PCRproducts. Finally, Pfu DNA polymerase is used to polish the ends of thePCR product prior to intramolecular ligation using T4 DNA ligase.

A non-limiting example for the isolation of mutant polynucleotides isdescribed in detail as follows:

Plasmid template DNA (approximately 0.5 pmole) is added to a PCRcocktail containing: 1× mutagenesis buffer (20 mM Tris HCl, pH 7.5; 8 mMMgCl2; 40 g/ml BSA); 12-20 pmole of each primer (one of skill in the artmay design a mutagenic primer as necessary, giving consideration tothose factors such as base composition, primer length and intendedbuffer salt concentrations that affect the annealing characteristics ofoligonucleotide primers; one primer must contain the desired mutation,and one (the same or the other) must contain a 5′ phosphate tofacilitate later ligation), 250 M each dNTP, 2.5 U Taq DNA polymerase,and 2.5 U of Taq Extender (Available from Stratagene; See Nielson et al.(1994) Strategies 7: 27, and U.S. Pat. No. 5,556,772). Primers can beprepared using the triester method of Matteucci et al., 1981, J. Am.Chem. Soc. 103:3185-3191, incorporated herein by reference.Alternatively automated synthesis may be preferred, for example, on aBiosearch 8700 DNA Synthesizer using cyanoethyl phosphoramiditechemistry.

The PCR cycling is performed as follows: 1 cycle of 4 min at 94° C., 2min at 50° C. and 2 min at 72° C.; followed by 5-10 cycles of 1 min at94° C., 2 min at 54° C. and 1 min at 72° C. The parental template DNAand the linear, PCR-generated DNA incorporating the mutagenic primer aretreated with DpnI (10 U) and Pfu DNA polymerase (2.5 U). This results inthe DpnI digestion of the in vivo methylated parental template andhybrid DNA and the removal, by Pfu DNA polymerase, of thenon-template-directed Taq DNA polymerase-extended base(s) on the linearPCR product. The reaction is incubated at 37° C. for 30 min and thentransferred to 72° C. for an additional 30 min. Mutagenesis buffer (115ul of 1×) containing 0.5 mM ATP is added to the DpnI-digested, Pfu DNApolymerase-polished PCR products. The solution is mixed and 10 ul areremoved to a new microfuge tube and T4 DNA ligase (2-4 U) is added. Theligation is incubated for greater than 60 min at 37° C. Finally, thetreated solution is transformed into competent E. coli according tostandard methods.

Methods of random mutagenesis, which will result in a panel of mutantsbearing one or more randomly situated mutations, exist in the art. Sucha panel of mutants may then be screened for those exhibiting reduceduracil detection activity relative to the wild-type polymerase (e.g., bymeasuring the incorporation of 10 nmoles of dNTPs into polymeric form in30 minutes in the presence of 200 M dUTP and at the optimal temperaturefor a given DNA polymerase). An example of a method for randommutagenesis is the so-called “error-prone PCR method”. As the nameimplies, the method amplifies a given sequence under conditions in whichthe DNA polymerase does not support high fidelity incorporation. Theconditions encouraging error-prone incorporation for different DNApolymerases vary, however one skilled in the art may determine suchconditions for a given enzyme. A key variable for many DNA polymerasesin the fidelity of amplification is, for example, the type andconcentration of divalent metal ion in the buffer. The use of manganeseion and/or variation of the magnesium or manganese ion concentration maytherefore be applied to influence the error rate of the polymerase.

Genes for desired mutant polypeptides generated by mutagenesis may besequenced to identify the sites and number of mutations. For thosemutants comprising more than one mutation, the effect of a givenmutation may be evaluated by introduction of the identified mutation tothe wild-type gene by site-directed mutagenesis in isolation from theother mutations borne by the particular mutant. Screening assays of thesingle mutant thus produced will then allow the determination of theeffect of that mutation alone.

V. Genes and Proteins for the Production of ECO-04601

As discussed in more detail below, the isolated, purified or enrichednucleic acids of one of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 may beused to prepare one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84,86 and 88, respectively, or fragments comprising at least 50, 75, 100,200, 300, 500 or more consecutive amino acids of one of the polypeptidesof SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67,69, 71, 74, 76, 78, 80, 82, 84, 86 and 88.

Accordingly, another aspect of the present invention is an isolated,purified or enriched nucleic acid which encodes one of the polypeptidesof SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67,69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising atleast 50, 75, 100, 150, 200, 300 or more consecutive amino acids of oneof the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88. Thecoding sequences of these nucleic acids may be identical to one of thecoding sequences of one of the nucleic acids of SEQ ID NOS: 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83,85, 87 and 89 or a fragment thereof, or may be different codingsequences which encode one of the polypeptides of SEQ ID NOS: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80,82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150,200, 300 consecutive amino acids of one of the polypeptides of SEQ IDNOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74,76, 78, 80, 82, 84, 86 and 88 as a result of the redundancy ordegeneracy of the genetic code. The genetic code is well known to thoseof skill in the art and can be obtained, for example, from Stryer,Biochemistry, 3^(rd) edition, W.H. Freeman & Co., New York.

The isolated, purified or enriched nucleic acid which encodes one of thepolypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 may include, butis not limited to: (1) only the coding sequences of one of SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77,79, 81, 83, 85, 87 and 89; (2) the coding sequences of SEQ ID NOS: 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79,81, 83, 85, 87 and 89 and additional coding sequences, such as leadersequences or proprotein; and (3) the coding sequences of SEQ ID NOS: 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79,81, 83, 85, 87 and 89 and non-coding sequences, such as non-codingsequences 5′ and/or 3′ of the coding sequence. Thus, as used herein, theterm “polynucleotide encoding a polypeptide” encompasses apolynucleotide that includes only coding sequence for the polypeptide aswell as a polynucleotide that includes additional coding and/ornon-coding sequence.

The invention relates to polynucleotides based on SEQ ID NOS: 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81,83, 85, 87 and 89 but having polynucleotide changes that are “silent”,for example changes which do not alter the amino acid sequence encodedby the polynucleotides of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89. Theinvention also relates to polynucleotides which have nucleotide changeswhich result in amino acid substitutions, additions, deletions, fusionsand truncations of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86and 88. Such nucleotide changes may be introduced using techniques suchas site directed mutagenesis, random chemical mutagenesis, exonucleaseIII deletion, and other recombinant DNA techniques.

The isolated, purified or enriched nucleic acids of SEQ ID NOS: 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81,83, 85, 87 and 89, the sequences complementary thereto, or a fragmentcomprising at least 100, 150, 200, 300, 400 or more consecutive bases ofone of the sequence of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, or thesequences complementary thereto may be used as probes to identify andisolate DNAs encoding the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84,86 and 88 respectively. In such procedures, a genomic DNA library isconstructed from a sample microorganism or a sample containing amicroorganism capable of producing a farnesyl dibenzodiazepinone. Thegenomic DNA library is then contacted with a probe comprising a codingsequence or a fragment of the coding sequence, encoding one of thepolypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88, or a fragmentthereof under conditions which permit the probe to specificallyhybridize to sequences complementary thereto. In a preferred embodiment,the probe is an oligonucleotide of about 10 to about 30 nucleotides inlength designed based on a nucleic acid of SEQ ID NOS: 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85,87 and 89. Genomic DNA clones which hybridize to the probe are thendetected and isolated. Procedures for preparing and identifying DNAclones of interest are disclosed in Ausubel et al., Current Protocols inMolecular Biology, John Wiley 503 Sons, Inc. 1997; and Sambrook et al.,Molecular Cloning: A Laboratory Manual 2d Ed., Cold Spring HarborLaboratory Press, 1989. In another embodiment, the probe is arestriction fragment or a PCR amplified nucleic acid derived from SEQ IDNOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75,77, 79, 81, 83, 85, 87 and 89.

The isolated, purified or enriched nucleic acids of SEQ ID NOS: 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81,83, 85, 87 and 89, the sequences complementary thereto, or a fragmentcomprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200,300, 400 or 500 consecutive bases of one of the sequences of SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77,79, 81, 83, 85, 87 and 89 or the sequences complementary thereto may beused as probes to identify and isolate related nucleic acids. In someembodiments, the related nucleic acids may be genomic DNAs (or cDNAs)from potential farnesyl dibenzodiazepinone producers. In suchprocedures, a nucleic acid sample containing nucleic acids from apotential farnesyl dibenzodiazepinone producer is contacted with theprobe under conditions that permit the probe to specifically hybridizeto related sequences. The nucleic acid sample may be a genomic DNA (orcDNA) library from the potential farnesyl dibenzodiazepinone-producer.Hybridization of the probe to nucleic acids is then detected using anyof the methods described above.

Hybridization may be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's, and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4-9×10⁸ cpm/ug) of ³²Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh1×SET at Tm-10° C. for the oligonucleotide probe where Tm is the meltingtemperature. The membrane is then exposed to autoradiographic film fordetection of hybridization signals.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as genomic DNAs or cDNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature of the probemay be calculated using the following formulas:

For oligonucleotide probes between 14 and 70 nucleotides in length themelting temperature (Tm) in degrees Celcius may be calculated using theformula: T_(m)=81.5+16.6(log [Na⁺])+0.41 (fraction G+C)−(600/N) where Nis the length of the oligonucleotide.

If the hybridization is carried out in a solution containing formamide,the melting temperature may be calculated using the equationTm=81.5+16.6(log [Na+])+0.41 (fraction G+C)−(0.63% formamide)−(600/N)where N is the length of the probe.

Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5%SDS, 0.1 mg/ml denatured fragmented salmon sperm DNA or 6×SSC,5×Denhardt's reagent, 0.5% SDS, 0.1 mg/ml denatured fragmented salmonsperm DNA, 50% formamide. The composition of the SSC and Denhardt'ssolutions are listed in Sambrook et al., supra.

Hybridization is conducted by adding the detectable probe to thehybridization solutions listed above. Where the probe comprises doublestranded DNA, it is denatured by incubating at elevated temperatures andquickly cooling before addition to the hybridization solution. It mayalso be desirable to similarly denature single stranded probes toeliminate or diminish formation of secondary structures oroligomerization. The filter is contacted with the hybridization solutionfor a sufficient period of time to allow the probe to hybridize to cDNAsor genomic DNAs containing sequences complementary thereto or homologousthereto. For probes over 200 nucleotides in length, the hybridizationmay be carried out at 15-25° C. below the Tm. For shorter probes, suchas oligonucleotide probes, the hybridization may be conducted at 5-10°C. below the Tm. Preferably, the hybridization is conducted in 6×SSC,for shorter probes. Preferably, the hybridization is conducted in 50%formamide containing solutions, for longer probes. All the foregoinghybridizations would be considered to be examples of hybridizationperformed under conditions of high stringency.

Following hybridization, the filter is washed for at least 15 minutes in2×SSC, 0.1% SDS at room temperature or higher, depending on the desiredstringency. The filter is then washed with 0.1×SSC, 0.5% SDS at roomtemperature (again) for 30 minutes to 1 hour. Nucleic acids which havehybridized to the probe are identified by conventional autoradiographyand non-radioactive detection methods.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na+ concentration of approximately1M. Following hybridization, the filter may be washed with 2×SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate stringency” conditions above 50° C. and “low stringency”conditions below 50° C. A specific example of “moderate stringency”hybridization conditions is when the above hybridization is conducted at55° C. A specific example of “low stringency” hybridization conditionsis when the above hybridization is conducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate stringency” conditions above 25% formamide and “lowstringency” conditions below 25% formamide. A specific example of“moderate stringency” hybridization conditions is when the abovehybridization is conducted at 30% formamide. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 10% formamide. Nucleic acids which have hybridized to theprobe are identified by conventional autoradiography and non-radioactivedetection methods.

The preceding methods may be used to isolate nucleic acids having atleast 97%, at least 95%, at least 90%, at least 85%, at least 80%, or atleast 70% sequence identity to a nucleic acid sequence selected from thegroup consisting of the sequences of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and89. The isolated nucleic acid may have a coding sequence that is anaturally occurring allelic variant of one of the coding sequencesdescribed herein. Such allelic variant may have a substitution, deletionor addition of one or more nucleotides when compared to the nucleicacids of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66,68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, or the sequencescomplementary thereto.

Additionally, the above procedures may be used to isolate nucleic acidswhich encode polypeptides having at least 99%, at least 95%, at least90%, at least 85%, at least 80%, or at least 70% identity to apolypeptide having the sequence of one of SEQ ID NOS: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84,86 and 88 or fragments comprising at least 50, 75, 100, 150, 200, 300consecutive amino acids thereof.

Another aspect of the present invention is an isolated or purifiedpolypeptide comprising the sequence of one of SEQ ID NOS: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82,84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200 or300 consecutive amino acids thereof. As discussed herein, suchpolypeptides may be obtained by inserting a nucleic acid encoding thepolypeptide into a vector such that the coding sequence is operablylinked to a sequence capable of driving the expression of the encodedpolypeptide in a suitable host cell. For example, the expression vectormay comprise a promoter, a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for modulating expression levels, an origin ofreplication and a selectable marker.

Promoters suitable for expressing the polypeptide or fragment thereof inbacteria include the E. coli lac or trp promoters, the lad promoter, thelacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter, thelambda P_(R) promoter, the lambda P_(L) promoter, promoters from operonsencoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), andthe acid phosphatase promoter. Fungal promoters include the α factorpromoter. Eukaryotic promoters include the CMV immediate early promoter,the HSV thymidine kinase promoter, heat shock promoters, the early andlate SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused.

Mammalian expression vectors may also comprise an origin of replication,any necessary ribosome binding sites, a polyadenylation site, splicedonors and acceptor sites, transcriptional termination sequences, and 5′flanking nontranscribed sequences. In some embodiments, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required nontranscribed genetic elements.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

In addition, the expression vectors preferably contain one or moreselectable marker genes to permit selection of host cells containing thevector. Examples of selectable markers that may be used include genesencoding dihydrofolate reductase or genes conferring neomycin resistancefor eukaryotic cell culture, genes conferring tetracycline or ampicillinresistance in E. coli, and the S. cerevisiae TRP1 gene.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is ligated to thedesired position in the vector following digestion of the insert and thevector with appropriate restriction endonucleases. Alternatively,appropriate restriction enzyme sites can be engineered into a DNAsequence by PCR. A variety of cloning techniques are disclosed in Ausbelet al. Current Protocols in Molecular Biology, John Wiley 503 Sons, Inc.1997 and Sambrook et al., Molecular Cloning: A Laboratory Manual 2d Ed.,Cold Spring Harbour Laboratory Press, 1989. Such procedures and othersare deemed to be within the scope of those skilled in the art.

The vector may be, for example, in the form of a plasmid, a viralparticle, or a phage. Other vectors include derivatives of chromosomal,nonchromosomal and synthetic DNA sequences, viruses, bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, and pseudorabies. A variety of cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989).

Particular bacterial vectors which may be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), pGEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, phiX174, pBluescript™ II KS, pNH8A, pNH16a, pNH18A,pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia), pKK232-8 and pCM7. Particular eukaryotic vectors includepSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL(Pharmacia). However, any other vector may be used as long as it isreplicable and stable in the host cell.

The host cell may be any of the host cells familiar to those skilled inthe art, including prokaryotic cells or eukaryotic cells. Asrepresentative examples of appropriate hosts, there may be mentioned:bacteria cells, such as E. coli, Streptomyces lividans, Streptomycesgriseofuscus, Streptomyces ambofaciens, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, Bacillus, and Staphylococcus, fungal cells, such as yeast,insect cells such as Drosophila S2 and Spodoptera Sf9, animal cells suchas CHO, COS or Bowes melanoma, and adenoviruses. The selection of anappropriate host is within the abilities of those skilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including electroporation transformation, transfection,transduction, viral infection, gene guns, or Ti-mediated gene transfer.Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of thepresent invention. Following transformation of a suitable host strainand growth of the host strain to an appropriate cell density, theselected promoter may be induced by appropriate means (e.g., temperatureshift or chemical induction) and the cells may be cultured for anadditional period to allow them to produce the desired polypeptide orfragment thereof.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract is retained forfurther purification. Microbial cells employed for expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents. Such methods are well known to those skilled in the art.The expressed polypeptide or fragment thereof can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the polypeptide. Ifdesired, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts (described by Gluzman,Cell, 23:175 (1981)), and other cell lines capable of expressingproteins from a compatible vector, such as the C127, 3T3, CHO, HeLa andBHK cell lines. The constructs in host cells can be used in aconventional manner to produce the gene product encoded by therecombinant sequence. Polypeptides of the invention may or may not alsoinclude an initial methionine amino acid residue.

Alternatively, the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and88 or fragments comprising at least 50, 75, 100, 150, 200 or 300consecutive amino acids thereof can be synthetically produced byconventional peptide synthesizers. In other embodiments, fragments orportions of the polynucleotides may be employed for producing thecorresponding full-length polypeptide by peptide synthesis; therefore,the fragments may be employed as intermediates for producing thefull-length polypeptides.

Cell-free translation systems can also be employed to produce one of thepolypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragmentscomprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acidsthereof using mRNAs transcribed from a DNA construct comprising apromoter operably linked to a nucleic acid encoding the polypeptide orfragment thereof. In some embodiments, the DNA construct may belinearized prior to conducting an in vitro transcription reaction. Thetranscribed mRNA is then incubated with an appropriate cell-freetranslation extract, such as a rabbit reticulocyte extract, to producethe desired polypeptide or fragment thereof.

The present invention also relates to variants of the polypeptides ofSEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69,71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least50, 75, 100, 150, 200 or 300 consecutive amino acids thereof. The term“variant” includes derivatives or analogs of these polypeptides. Inparticular, the variants may differ in amino acid sequence from thepolypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 by one or moresubstitutions, additions, deletions, fusions and truncations, which maybe present in any combination.

The variants may be naturally occurring or created in vitro. Inparticular, such variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures.

Other methods of making variants are also familiar to those skilled inthe art. These include procedures in which nucleic acid sequencesobtained from natural isolates are modified to generate nucleic acidsthat encode polypeptides having characteristics which enhance theirvalue in industrial or laboratory applications. In such procedures, alarge number of variant sequences having one or more nucleotidedifferences with respect to the sequence obtained from the naturalisolate are generated and characterized. Preferably, these nucleotidedifferences result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

The variants of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and88 may be variants in which one or more of the amino acid residues ofthe polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code.

Conservative substitutions are those that substitute a given amino acidin a polypeptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the followingreplacements: replacements of an aliphatic amino acid such as Ala, Val,Leu and IIe with another aliphatic amino acid; replacement of a Ser witha Thr or vice versa; replacement of an acidic residue such as Asp or Gluwith another acidic residue; replacement of a residue bearing an amidegroup, such as Asn or Gln, with another residue bearing an amide group;exchange of a basic residue such as Lys or Arg with another basicresidue; and replacement of an aromatic residue such as Phe or Tyr withanother aromatic residue.

Other variants are those in which one or more of the amino acid residuesof the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 include asubstituent group. Still other variants are those in which thepolypeptide is associated with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol). Additional variants are those in which additional amino acidsare fused to the polypeptide, such as leader sequence, a secretorysequence, a proprotein sequence or a sequence that facilitatespurification, enrichment, or stabilization of the polypeptide.

In some embodiments, the fragments, derivatives and analogs retain thesame biological function or activity as the polypeptides of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76,78, 80, 82, 84, 86 and 88. In other embodiments, the fragment,derivative or analogue includes a fused heterologous sequence thatfacilitates purification, enrichment, detection, stabilization orsecretion of the polypeptide that can be enzymatically cleaved, in wholeor in part, away from the fragment, derivative or analogue.

Another aspect of the present invention are polypeptides or fragmentsthereof which have at least 70%, at least 80%, at least 85%, at least90%, or more than 95% identity to one of the polypeptides of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76,78, 80, 82, 84, 86 and 88 or a fragment comprising at least 50, 75, 100,150, 200 or 300 consecutive amino acids thereof. It will be appreciatedthat amino acid “substantially identity” includes conservativesubstitutions such as those described above.

The polypeptides or fragments having homology to one of the polypeptidesof SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67,69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or a fragment comprising atleast 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof maybe obtained by isolating the nucleic acids encoding them using thetechniques described above.

Alternatively, the homologous polypeptides or fragments may be obtainedthrough biochemical enrichment or purification procedures. The sequenceof potentially homologous polypeptides or fragments may be determined byproteolytic digestion, gel electrophoresis and/or microsequencing. Thesequence of the prospective homologous polypeptide or fragment can becompared to one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86and 88 or a fragment comprising at least 5, 10, 15, 20, 25, 30, 35, 40,50, 75, 100, or 150 consecutive amino acids thereof.

The polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments,derivatives or analogs thereof comprising at least 40, 50, 75, 100, 150,200 or 300 consecutive amino acids thereof invention may be used in avariety of applications. For example, the polypeptides or fragments,derivatives or analogs thereof may be used to catalyze biochemicalreactions as described elsewhere in the specification.

(a)

VI. Pharmaceutical Compositions Comprising Farnesyl Dibenzodiazepinones

In another embodiment, the invention relates to a pharmaceuticalcomposition comprising a farnesyl dibenzodiazepinone, as described inthe preceding section, and a pharmaceutically acceptable carrier, asdescribed below. The pharmaceutical composition comprising the farnesyldibenzodiazepinone is useful for treating a variety of diseases anddisorders, including cancer, inflammation and bacterial infections.

The compounds of the present invention, or pharmaceutically acceptablesalts thereof, can be formulated for oral, intravenous, intramuscular,subcutaneous, topical or parenteral administration for the therapeuticor prophylactic treatment of diseases, particularly bacterialinfections, acute and chronic inflammation and cancer. For oral orparental administration, compounds of the present invention can be mixedwith conventional pharmaceutical carriers and excipients and used in theform of tablets, capsules, elixirs, suspensions, syrups, wafers and thelike. The compositions comprising a compound of this present inventionwill contain from about 0.1% to about 99.9%, about 1% to about 98%,about 5% to about 95%, about 10% to about 80% or about 15% to about 60%by weight of the active compound.

The pharmaceutical preparations disclosed herein are prepared inaccordance with standard procedures and are administered at dosages thatare selected to reduce, prevent, or eliminate bacterial infection,cancer or inflammation. (See, e.g., Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa.; and Goodman and Gilman,Pharmaceutical Basis of Therapeutics, Pergamon Press, New York, N.Y.,the contents of which are incorporated herein by reference, for ageneral description of the methods for administering variousantimicrobial agents for human therapy). The compositions of the presentinvention can be delivered using controlled (e.g., capsules) orsustained release delivery systems (e.g., bioerodable matrices).Exemplary delayed release delivery systems for drug delivery that aresuitable for administration of the compositions of the invention(preferably of Formula I) are described in U.S. Pat. Nos. 4,452,775(issued to Kent), 5,239,660 (issued to Leonard), 3,854,480 (issued toZaffaroni).

The pharmaceutically acceptable compositions of the present inventioncomprise one or more compounds of the present invention in associationwith one or more non-toxic, pharmaceutically acceptable carriers and/ordiluents and/or adjuvants and/or excipients, collectively referred toherein as “carrier” materials, and if desired other active ingredients.The compositions may contain common carriers and excipients, such ascorn starch or gelatin, lactose, sucrose, microcrystalline cellulose,kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid.The compositions may contain crosarmellose sodium, microcrystallinecellulose, sodium starch glycolate and alginic acid.

Tablet binders that can be included are acacia, methylcellulose, sodiumcarboxymethylcellulose, polyvinylpyrrolidone (Providone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose.

Lubricants that can be used include magnesium stearate or other metallicstearates, stearic acid, silicon fluid, talc, waxes, oils and colloidalsilica.

Flavouring agents such as peppermint, oil of wintergreen, cherryflavouring or the like can also be used. It may also be desirable to adda coloring agent to make the dosage form more aesthetic in appearance orto help identify the product comprising a compound of the presentinvention.

For oral use, solid formulations such as tablets and capsules areparticularly useful. Sustained released or enterically coatedpreparations may also be devised. For pediatric and geriatricapplications, suspension, syrups and chewable tablets are especiallysuitable. For oral administration, the pharmaceutical compositions arein the form of, for example, a tablet, capsule, suspension or liquid.The pharmaceutical composition is preferably made in the form of adosage unit containing a therapeutically-effective amount of the activeingredient. Examples of such dosage units are tablets and capsules. Fortherapeutic purposes, the tablets and capsules which can contain, inaddition to the active ingredient, conventional carriers such as bindingagents, for example, acacia gum, gelatin, polyvinylpyrrolidone,sorbitol, or tragacanth; fillers, for example, calcium phosphate,glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, forexample, magnesium stearate, polyethylene glycol, silica or talc:disintegrants, for example, potato starch, flavoring or coloring agents,or acceptable wetting agents. Oral liquid preparations generally are inthe form of aqueous or oily solutions, suspensions, emulsions, syrups orelixirs and may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous agents, preservatives, coloringagents and flavoring agents. Examples of additives for liquidpreparations include acacia, almond oil, ethyl alcohol, fractionatedcoconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats,lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate,propylene glycol, sorbitol, or sorbic acid.

For intravenous (iv) use, compounds of the present invention can bedissolved or suspended in any of the commonly used intravenous fluidsand administered by infusion. Intravenous fluids include, withoutlimitation, physiological saline or Ringer's solution.

Formulations for parental administration can be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions or suspensions can be prepared from sterile powders orgranules having one or more of the carriers mentioned for use in theformulations for oral administration. The compounds can be dissolved inpolyethylene glycol, propylene glycol, ethanol, corn oil, benzylalcohol, sodium chloride, and/or various buffers.

For intramuscular preparations, a sterile formulation of compounds ofthe present invention or suitable soluble salts forming the compound,can be dissolved and administered in a pharmaceutical diluent such asWater-for-Injection (WFI), physiological saline or 5% glucose. Asuitable insoluble form of the compound may be prepared and administeredas a suspension in an aqueous base or a pharmaceutically acceptable oilbase, e.g. an ester of a long chain fatty acid such as ethyl oleate.

For topical use the compounds of present invention can also be preparedin suitable forms to be applied to the skin, or mucus membranes of thenose and throat, and can take the form of creams, ointments, liquidsprays or inhalants, lozenges, or throat paints. Such topicalformulations further can include chemical compounds such asdimethylsulfoxide (DMSO) to facilitate surface penetration of the activeingredient.

For application to the eyes or ears, the compounds of the presentinvention can be presented in liquid or semi-liquid form formulated inhydrophobic or hydrophilic bases as ointments, creams, lotions, paintsor powders.

For rectal administration the compounds of the present invention can beadministered in the form of suppositories admixed with conventionalcarriers such as cocoa butter, wax or other glyceride.

Alternatively, the compound of the present invention can be in powderform for reconstitution in the appropriate pharmaceutically acceptablecarrier at the time of delivery. In another embodiment, the unit dosageform of the compound can be a solution of the compound or a salt thereofin a suitable diluent in sterile, hermetically sealed ampoules.

The amount of the compound of the present invention in a unit dosagecomprises a therapeutically-effective amount of at least one activecompound of the present invention which may vary depending on therecipient subject, route and frequency of administration. A recipientsubject refers to a plant, a cell culture or an animal such as an ovineor a mammal including a human.

According to this aspect of the present invention, the novelcompositions disclosed herein are placed in a pharmaceuticallyacceptable carrier and are delivered to a recipient subject (including ahuman subject) in accordance with known methods of drug delivery. Ingeneral, the methods of the invention for delivering the compositions ofthe invention in vivo utilize art-recognized protocols for deliveringthe agent with the only substantial procedural modification being thesubstitution of the compounds of the present invention for the drugs inthe art-recognized protocols.

Likewise, the methods for using the claimed composition for treatingcells in culture, for example, to eliminate or reduce the level ofbacterial contamination of a cell culture, utilize art-recognizedprotocols for treating cell cultures with antibacterial agent(s) withthe only substantial procedural modification being the substitution ofthe compounds of the present invention for the agents used in theart-recognized protocols.

The compounds of the present invention provide a method for treatingbacterial infections, pre-cancerous or cancerous conditions, and acuteor chronic inflammatory disease. As used herein, the term “unit dosage”refers to a quantity of a therapeutically effective amount of a compoundof the present invention that elicits a desired therapeutic response. Asused herein, the phrase “therapeutically effective amount” means anamount of a compound of the present invention that prevents the onset,alleviates the symptoms, or stops the progression of a bacterialinfection, inflammatory condition, or pre-cancerous or cancerouscondition. The term “treating” is defined as administering, to asubject, a therapeutically effective amount of at least one compound ofthe present invention, both to prevent the occurrence of a bacterialinfection, inflammation or pre-cancer or cancer condition, or to controlor eliminate a bacterial infection, inflammation or pre-cancer or cancercondition. The term “desired therapeutic response” refers to treating arecipient subject with a compound of the present invention such that abacterial or inflammatory condition or pre-cancer or cancer condition isreversed, arrested or prevented in a recipient subject.

The compounds of the present invention can be administered as a singledaily dose or in multiple doses per day. The treatment regime mayrequire administration over extended periods of time, e.g., for severaldays or for from two to four weeks. The amount per administered dose orthe total amount administered will depend on such factors as the natureand severity of the disease condition, the age and general health of therecipient subject, the tolerance of the recipient subject to thecompound and the type of the bacterial infection, inflammatory disorder,or type of cancer.

A compound according to this invention may also be administered in thediet or feed of a patient or animal. The diet for animals can be normalfoodstuffs to which the compound can be added or it can be added to apremix.

The compounds of the present invention may be taken in combination,together or separately with any known clinically approved antibiotic,inflammation or anti-cancer agent to treat a recipient subject in needof such treatment.

VII. Method of Inhibiting Tumor Growth

In another embodiment, the present invention relates to a method ofinhibiting tumor growth. Compounds as described herein can possessantitumor activity. The compounds are effective against mammalian tumorcells such as leukemia cells, melanoma cells, breast carcinoma cells,lung carcinoma cells, pancreatic carcinoma cells, ovarian carcinomacells, renal carcinoma cells, colon carcinoma cells prostate carcinomacells and glioma cells. The antitumor method of the invention results ininhibition of tumor cells. The term “inhibition”, when used inconjunction with the antitumor method refers to suppression, killing,stasis, or destruction of tumor cells. The antitumor method preferablyresults in prevention, reduction or elimination of invasive activity andrelated metastasis of tumor cells. The term “effective amount” when usedin conjunction with the antitumor cell method refers to the amount ofthe compound sufficient to result in the inhibition of mammalian tumorcells.

The inhibition of mammalian tumor growth according to this method can bemonitored in several ways. First, tumor cells grown in vitro can betreated with the compound and monitored for growth or death relative tothe same cells cultured in the absence of the compound. A cessation ofgrowth or a slowing of the growth rate (i.e., the doubling rate), e.g.,by 10% or more, is indicative of tumor cell inhibition. Alternatively,tumor cell inhibition can be monitored by administering the compound toan animal model of the tumor of interest. Examples of experimentalanimal tumor models are known in the art and described in the examplesherein. A cessation of tumor growth (i.e., no further increase in size)or a reduction in tumor size (i.e., tumor volume) or cell number (e.g.,at least a 10% decrease in either) in animals treated with a compound asdescribed herein relative to tumors in control animals not treated withthe compound is indicative of tumor growth inhibition.

To monitor the efficacy of tumor treatment in a human, tumor size ortumor cell titer is measured before and after initiation of thetreatment, and treatment is considered effective if either the tumorsize or titer ceases further growth, or if the tumor is reduced in sizeor titer, e.g., by at least 10% or more (e.g., 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or even 100%, that is, the absence of the tumor). Methodsof determining the size or cell titer of a tumor in vivo vary with thetype of tumor, and include, for example, various imaging techniques wellknown to those in the medical imaging or oncology fields (MRI, CAT, PET,etc.), as well as histological techniques and flow cytometry.

For the antitumor method of the invention, a typical effective dose ofthe compounds given orally or parenterally would be from about 5 toabout 100 mg/kg of body weight of the subject with a daily dose rangingfrom about 15 to about 300 mg/kg of body weight of the subject.

VIII. Method of Inhibiting Lipoxygenase

In another embodiment, the present invention also provides for a methodof treating diseased states, in particular inflammation, caused by the5-lipoxygenase system and/or by the synthesis of the Leukotrienes C₄,D₄, E₄ and F₄ as well as Leukotriene B₄ in mammals, especially in humansubjects. This method comprises administering to a subject an effectiveamount of ECO-04601. Compound ECO-04601 may be used alone or incombination with other anti-inflammatory compounds to treat or preventdisease states related to inflammation including pulmonary conditions,inflammation, cardiovascular conditions, central nervous systemconditions or skin conditions. More specific diseases include gastritis;erosive esophagitis; inflammatory bowel disease; ethanol-inducedhemorrhagic erosions; hepatic ischemia; ischemic neuronal injury;noxious agent induced damage or necrosis of hepatic, pancreatic, renal,neuronal or myocardial tissue; liver parenchymal damage caused byhepatoxic agents such as CCl₄ and D-galactosamine; ischemic renalfailure; disease-induced hepatic damage; trauma- or stress-induced celldamage; asthma; multiple sclerosis; ischemic reperfusion; edema;rheumatoid arthritis; viral encephalitis; bacterial pneumonia;neurodegeneration; Alzheimer's disease and glycerol-induced renalfailure.

For the method of the invention related to the 5-lipoxygenase systemand/or the biosynthesis of Leukotrienes, a typical effective unit doseof ECO-04601 given orally or parenterally would be from about 5 to about100 mg/kg of body weight of the subject with a daily dose ranging fromabout 15 to about 300 mg/kg of body weight of the subject.

The inhibition of lipoxygenase enzymes is monitored using methods wellknown in the art and as described in the examples herein. A decrease inenzyme activity by at least 10%, relative to the activity in the absenceof a compound as described herein is indicative of effective inhibitionof lipoxygenase activity.

Farnesyl dibenzodiazepinone compounds useful according to the inventioncan be used to reduce or prevent inflammation. Among the hallmarks oflocal acute inflammation are heat, redness, swelling, pain and loss offunction. These changes are induced largely by changes in vascular flowand caliber, changes in vascular permeability and leukocyte exudation(Robbins et al., “Pathologic Basis of Disease”, 6^(th) Ed., W.B.Saunders Co., Philadelphia, Pa.). Anti-inflammatory therapy performedusing compounds useful according to the invention can be monitored forsuccess by tracking any of these changes. For example, a decrease inswelling (e.g., at least 10% decrease following treatment) or reportedpain (e.g., a sustained decrease of 1 point or more on a 1-10 scalereported by the patient, with 10 being the worst pain experienced inassociation with this disorder prior to treatment, and 0 being no pain)can be used to indicate successful treatment.

Other measurable hallmarks of inflammation include leukocyteinfiltration and inflammatory cytokine levels. These hallmarks can bemonitored by biopsy of the affected tissue. A decrease of 10% or more inleukocyte infiltration in fixed, stained tissue relative to infiltrationin similar tissue prior to treatment can be used to indicate successfultreatment, as can a decrease of 10% or more in the level of any giveninflammatory cytokine, relative to the level before treatment. Thoseskilled in the art can readily assay for inflammatory cytokine levels intissue, blood, or other fluid samples. Alternatively, the level ofsystemic indicators of inflammation such as C reactive protein levelsand erythrocyte sedimentation rate can be monitored. Each of these hasestablished normal ranges in medicine, and treatment is consideredsuccessful if one or more of such indicators goes from outside thenormal range to inside the normal range after the initiation oftreatment.

IX. Method of Inhibiting Bacterial Growth

In another embodiment, the present invention relates to a method fortreating bacterial infection in a mammalian subject in need thereof,comprising the step of administering to the mammal a therapeuticallyeffective amount of compound ECO-04601, a compound as described herein,or a pharmaceutically acceptable derivative or prodrug thereof.

According to another embodiment, the invention provides a method ofdecreasing bacterial quantity in a biological sample. This methodcomprises the step of contacting the biological sample with a compoundECO-04601, a compound as described herein, or a pharmaceuticallyacceptable derivative or prodrug thereof. This method is effective ifthe number of bacteria decreases by at least 10%, and preferably more,e.g., 25%, 50%, 75% or even 100% after contacting the biological samplewith compound ECO-04601, a compound as described herein, or apharmaceutically acceptable derivative or prodrug thereof.

These pharmaceutical compositions effective to treat or prevent abacterial infection which comprise ECO-04601, a compound as describedherein, or a pharmaceutically acceptable derivative or prodrug thereofin an amount sufficient to measurably decrease bacterial quantity, and apharmaceutically acceptable carrier, are another embodiment of thepresent invention. The term “measurably decrease bacterial quantity”, asused herein means a measurable change in the number of bacteria betweena sample containing the inhibitor and a sample not containing theinhibitor.

Agents which increase the susceptibility of bacterial organisms toantibiotics are known. For example, U.S. Pat. No. 5,523,288, U.S. Pat.No. 5,783,561 and U.S. Pat. No. 6,140,306 describe methods of usingbactericidal/permeability-increasing protein (BPI) for increasingantibiotic susceptibility of gram-positive and gram-negative bacteria.Agents that increase the permeability of the outer membrane of bacterialorganisms have been described by Vaara, M. in Microbiological Reviews(1992) pp. 395-411, and the sensitization of gram-negative bacteria hasbeen described by Tsubery, H., et al, in J. Med. Chem. (2000) pp.3085-3092.

For the method of the invention related to treatment of subjects with abacterial infection, a typical effective unit dose of ECO-04601, acompound described herein or a pharmaceutically acceptable derivative orprodrug thereof given orally or parenterally would be from about 5 toabout 100 mg/kg of body weight of the subject with a daily dose rangingfrom about 15 to about 300 mg/kg of body weight of the subject.

Another preferred embodiment of this invention relates to a method, asdescribed above, of treating a bacterial infection in a mammal in needthereof, but further comprising the step of administering to the mammalan agent which increases the susceptibility of bacterial organisms toantibiotics.

According to another preferred embodiment, the invention provides amethod, as described above, of decreasing bacterial quantity in abiological sample, but further comprising the step of contacting thebiological sample with an agent which increases the susceptibility ofbacterial organisms to antibiotics.

Methods of decreasing bacterial quantity are effective if the number ofbacteria decreases at least 10%, and preferably more, e.g., 25%, 50%,75% or even 100% after contacting the biological sample with compoundECO-04601, a compound as described herein, or a pharmaceuticallyacceptable derivative or prodrug thereof.

The pharmaceutical compositions and methods of this invention will beuseful generally for controlling bacterial infections in vivo. Examplesof bacterial organisms that may be controlled by the compositions andmethods of this invention include, but are not limited to the followingorganisms: Streptococcus pneumoniae, Streptococcus pyrogenes,Enterococcus fecalis, Enterococcus faecium, Klebsiella pneumoniae,Enterobacter spp., Proteus spp., Pseudomonas aeruginosa, E. coli,Serratia marcesens, Staphylococcus aureus, Coagulase negativeStaphylococcus, Haemophilus infuenzae, Bacillus anthracis, Mycoplasmapneumoniae, and Staphylococcus epidermidis. The compositions and methodswill therefore be useful for controlling, treating or reducing theadvancement, severity or effects of nosocomial or non-nosocomialinfections. Examples of nosocomial uses include, but are not limited to,urinary tract infections, pneumonia, surgical wound infections,bacteremia and therapy for febrile neutropenic patients. Examples ofnon-nosocomial uses include but are not limited to urinary tractinfections, pneumonia, prostatitis, skin and soft tissue infections andintra-abdominal infections.

In addition to the compounds of this invention, pharmaceuticallyacceptable derivatives or prodrugs of the compounds of this inventionmay also be employed in compositions to treat or prevent theabove-identified disorders.

A “pharmaceutically acceptable derivative or prodrug” means anypharmaceutically acceptable salt, ester, salt of an ester or otherderivative of a compound of this invention which, upon administration toa recipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof. Particularly favored derivatives or prodrugs are thosethat increase the bioavailability of the compounds of this inventionwhen such compounds are administered to a mammal (e.g., by allowing anorally administered compound to be more readily absorbed into the blood)or which enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) relative to the parentspecies.

Pharmaceutically acceptable prodrugs of the compounds of this inventioninclude, without limitation, esters, amino acid esters, phosphateesters, metal salts and sulfonate esters.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,IC₅₀ and so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about”.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the present specification and attached claims areapproximations. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of significant figures and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set in the examples, Tables and Figures are reported asprecisely as possible. Any numerical values may inherently containcertain errors resulting from variations in experiments, testingmeasurements, statistical analyses and such.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES Example 1 Preparation of Production Culture

Unless otherwise noted, all reagents were purchased from Sigma ChemicalCo. (St. Louis, Mo.), (Aldrich). Micromonospora spp. (deposit accessionnumber IDAC 070303-01) was maintained on agar plates of ISP2 agar (DifcoLaboratories, Detroit, Mich.). An inoculum for the production phase wasprepared by transferring the surface growth of the Micromonospora spp.from the agar plates to 125-mL flasks containing 25 mL of sterile mediumcomprised of 24 g potato dextrin, 3 g beef extract, 5 g Bacto-casitone,5 g glucose, 5 g yeast extract, and 4 g CaCO₃ made up to one liter withdistilled water (pH 7.0). The culture was incubated at about 28° C. forapproximately 60 hours on a rotary shaker set at 250 rpm. Followingincubation, 10 mL of culture was transferred to a 2 L baffled flaskcontaining 500 mL of sterile production medium containing 20 g/L potatodextrin, 20 g/L glycerol, 10 g/L Fish meal, 5 g/L Bacto-peptone, 2 g/LCaCO₃, and 2 g/L (NH₄)₂SO₄, pH 7.0. Fermentation broth was prepared byincubating the production culture at 28° C. in a rotary shaker set at250 rpm for one week.

Example 2 Isolation

500 mL ethyl acetate was added to 500 mL of fermentation broth preparedas described in Example 1 above. The mixture was agitated for 30 minuteson an orbital shaker at 200 rpm to create an emulsion. The phases wereseparated by centrifugation and decantation. Between 4 and 5 g ofanhydrous MgSO₄ was added to the organic phase, which was then filteredand the solvents removed in vacuo.

An ethyl acetate extract from 2 L fermentation was mixed with HP-20resin (100 mL; Mitsubishi Casei Corp., Tokyo, Japan) in water (300 mL).Ethyl acetate was removed in vacuo, the resin was filtered on a Buchnerfunnel and the filtrate was discarded. The adsorbed HP-20 resin was thenwashed successively with 2×125 mL of 50% acetonitrile in water, 2×125 mLof 75% acetonitrile in water and 2×125 mL of acetonitrile.

Fractions containing the compound of Formula II were evaporated todryness and 100 mg was digested in the 5 mL of the upper phase of amixture prepared from chloroform, cyclohexane, methanol, and water inthe ratios, by volume, of 5:2:10:5. The sample was subjected tocentrifugal partition chromatography using a High Speed Countercurrent(HSCC) system (Kromaton Technologies, Angers, France) fitted with a 200mL cartridge and prepacked with the upper phase of this two-phasesystem. The HSCC was run with the lower phase mobile and the compound ofFormula II was eluted at approximately one-half column volume. Fractionswere collected and the compound of Formula II was detected by TLC ofaliquots of the fractions on commercial Kieselgel 60F₂₅₄ plates.Compound could be visualized by inspection of dried plates under UVlight or by spraying the plates with a spray containing vanillin (0.75%)and concentrated sulfuric acid (1.5%, v/v) in ethanol and subsequentlyheating the plate. Fractions contained substantially pure compound ofFormula II, although highly colored. A buff-colored sample could beobtained by chromatography on HPLC as follows.

6 mg of sample was dissolved in acetonitrile and injected onto apreparative HPLC column (XTerra ODS (10 μm), 19×150 mm, Waters Co.,Milford, Mass.), with a 9 mL/min flow rate and UV peak detection at 300nm. The column was eluted with acetonitrile/buffer (20 mM of NH₄HCO₃)according to the following gradient shown in Table 1

TABLE 1 Time (min) Water (%) Acetonitrile (%) 0 70 30 10 5 95 15 5 95 2070 30

Fractions containing the compound of Formula II eluted at approximately11:0 min and were combined, concentrated and lyophilized to give a yieldof 3.8 mg compound.

Alternative Protocol 1

The compound of Formula II was also isolated using the followingalternative protocol. At the end of the incubation period, thefermentation broth from the baffled flasks of Example 1 was centrifugedand the supernatant decanted from the pellet containing the bacterialmycelia. 100 mL of 100% MeOH was added to the mycelial pellet and thesample was stirred for 10 minutes and centrifuged for 15 minutes. Themethanolic supernatant was decanted and saved. 100 mL of acetone wasthen added to the mycelial pellet and stirred for 10 minutes thencentrifuged for 15 minutes. The acetonic supernatant was decanted andcombined with the methanolic supernatant. Finally, 100 mL of 20%MeOH/H₂O was added to the mycelial pellet, stirred for 10 minutes andcentrifuged for 15 minutes. The supernatant was combined with theacetonic and methanolic supernatants.

The combined supernatant was added to 400 ml of HP-20 resin in 1000 mLof water and the organics were removed in vacuo. The resulting slurrywas filtered on a Buchner funnel and the filtrate was discarded.Adsorbed HP-20 resin was washed successively with 2×500 mL of 50%MeOH/H₂O, 2×500 mL of 75% MeOH/H₂O and 2×500 mL of MeOH.

The individual washes were collected separately and analyzed by TLC asdescribed above. Those fractions containing the compound of Formula IIwere evaporated to near dryness and lyophilized. The lyophilizate wasdissolved in methanol and injected onto a preparative HPLC column(Xterra ODS (10 μm), 19×150 mm, Waters Co., Milford, Mass.) with a flowrate of 9 mL/min and peak detection at 300 nm.

The column was eluted with acetonitrile/buffer (5 mM of NH₄HCO₃)according to gradient shown in Table 2.

TABLE 2 Time (min) Buffer (%) Acetonitrile (%) 0 95 5 15 45 55 20 5 9530 5 95 35 95 5

Fractions containing the compound of Formula II were combined,concentrated and lyophilized to yield about 33.7 mg of compound.

Alternative Protocol 2

10 liters of the whole broth from Example 1 are extracted twice withequal volumes of ethyl acetate and the two extracts are combined andconcentrated to dryness. The dried extract is weighed, and for everygram of dry extract, 100 mL of MeOH—H₂O (2:1 v/v) and 100 mL of hexaneis added. The mixture is swirled gently but well to achieve dissolution.The two layers are separated and the aqueous layer is washed with 100 mLof hexane. The two hexane layers are combined and the combined hexanesolution is washed with 100 mL methanol:water (2:1, v/v). The twomethanol:water layers are combined and treated with 200 mL of EtOAc and400 mL of water. The layers are separated and the aqueous layer isextracted twice more with 200 mL portions of EtOAc. The EtOAc layers arecombined and concentrated. The residue obtained will be suitable forfinal purification, either by HSCC or by HPLC as described above. Thisextraction process achieves a ten-fold purification when compared withthe extraction protocol used above.

Example 3 Elucidation of the Structure of Compound of Formula II

The structure of the compound of Formula II was derived fromspectroscopic data, including mass, UV, and NMR spectroscopy. Mass wasdetermined by electrospray mass spectrometry to be 462.6 (FIG. 1), UVmax230 nm with a shoulder at 290 nm (FIG. 2). NMR data were collecteddissolved in MeOH-d₄ including proton (FIG. 3), and multidimensionalpulse sequences gDQCOSY (FIG. 4), gHSQC (FIG. 5), gHMBC (FIG. 6), andNOESY (FIG. 7).

A number of cross peaks in the 2D spectra of ECO-04601 are key in thestructural determination. For example, the farnesyl chain is placed onthe amide nitrogen by a strong cross peak between the proton signal ofthe terminal methylene of that chain at 4.52 ppm and the amide carbonylcarbon at 170 ppm in the gHMBC experiment. This conclusion is confirmedby a cross peak in the NOESY spectrum between the same methylene signalsat 4.52 ppm and the aromatic proton signal at 6.25 ppm from one of thetwo protons of the tetra substituted benzenoid ring.

Based on the mass, UV and NMR spectroscopy data, the structure of thecompound was determined to be the structure of Formula II.

Example 4 Antibacterial Activity (Minimal Inhibitory ConcentrationDetermination)

Minimal Inhibitory Concentration (MIC) is defined as the lowestconcentration of drug that inhibits more than 99% of the bacterialpopulation. The MIC determination of ECO-04601 against bacteria strains(Bacillus subtilis—ATCC 23857; Micrococcus luteus —ATCC 9341) wasperformed using broth microdilution assay (Methods for DilutionAntimicrobial Susceptibility Tests for Bacteria That Grow Aerobically;Approved Standard-Fifth Edition. NCCLS document M7-A5 (ISBN1-56238-394-9). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pa.19087-1898 USA.).

Test compound preparation: The test article ECO-04601 is prepared as100× stock solutions in DMSO, with concentrations ranging from 3.2 mg/mlto 0.0625 mg/ml (a two-fold dilution series over 10 points). The firstdilution (3.2 mg/ml) was prepared by resuspending 0.5 mg of each testarticle in 156.25 μl of DMSO. The stock is then serially diluted bytwo-fold decrement to obtain the desired concentration range.Inoculum preparation: From an overnight culture in Mueller Hinton (MH)broth, cell density for each indicator strain (Bacillus subtilis;Micrococcus luteus) was adjusted to 0.5 Mc Farland units in 0.85%saline, then further diluted 1/100 in appropriate assay medium (˜1×10⁶cells/ml).MIC determination: The 100×ECO-04601 solutions was diluted 50 times inMH broth and dispensed in a 96 well plate, one test concentration percolumn of wells, 10 columns in total. The 11^(th) column of wellscontained MH broth with 1% DMSO, the 12^(th) column of wells contained100 μl of broth alone. 50 μl of the final cell dilution of eachindicator strain was added to each corresponding well of the microplatecontaining 50 μl of diluted drug or media alone. Assay plates wereincubated at 35° C. for 24 hrs.The results of the MIC for the compound of ECO-04601, shown in Table 3,demonstrate a range of antibacterial effects:

TABLE 3 Indicator strain MIC (μg/mL) Bacillus subtilis ATCC 23857 12.5Micrococcus luteus ATCC 9341 6.25

Example 5 Anticancer Activity In Vitro Against Human and Animal TumorCell Lines from Various Tissues

Culture conditions: The cell lines listed in Table 4 were used tocharacterize the cytotoxicity of ECO-04601 against human and animaltumor cell lines. These cell lines were shown to be free of mycoplasmainfection and were maintained on the appropriate media (Table 4)supplemented with 10% heat-inactivated fetal bovine serum and 1%penicillin-streptomycin, under 5% CO₂ at 37° C. Cells were passagedtwice to three times per week. Viability was examined by staining with0.25% trypan blue and only flasks where cell viability was >95% wereused for this study.Cell lines amplification and plating: Tumor cells were seeded (1-3×10³cells per 100 μL) in 96-wells flat bottom microtiter plates andincubated at 37° C. and 5% CO₂ for 16 hrs before treatment in drug-freemedium supplemented with 10% serum.Evaluation of inhibitory activity on cell proliferation: Cells wereincubated for 96 hrs with 6 log₁₀-fold concentrations of the testsubstance starting at 10 μg/ml (20 μM). The test substance stocksolution (5 mg/mL) was initially diluted at 1/70 fold in mediumsupplemented with serum. Other concentrations were then obtained from1/10 fold successive dilutions in the same supplemented medium. Cellsurvival was evaluated 96 h later by replacing the culture media with150 μL fresh medium containing 10 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4. Next,50 μL of 2.5 mg/mL of3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) inphosphate buffer solution, pH 7.4, was added. After 3-4 h of incubationat 37° C., the medium and soluble MTT was removed, and 200 μL ofdimethylsulfoxide was added to dissolve the precipitate of reduced MTTfollowed by addition of 25 μL glycine buffer (0.1 M glycine plus 0.1 MNaCl, pH 10.5). The absorbance was determined at 570 nm with amicroplate reader. Results were expressed as the concentration of drugwhich inhibits 50% of the cell growth (IC₅₀). The IC₅₀ values shown inTable 4 demonstrated a pharmacologically relevant cytotoxic activity ofECO-04601 against a variety of tumor types such as leukemias, melanomas,pancreatic and breast carcinomas.

TABLE 4 Cell Culture IC₅₀ lines Type Origin Source medium (x10⁻⁵ M) K562Leukemia Human ATCC RPMI 1640 8.6 myelogeneous P388 Leukemia Mouse ATCCRPMI 1640 10.9 I83 Leukemia Human ATCC RPMI 1640 2.7 B16 (F10) MelanomaMouse ATCC RPMI 1640 11.4 SK-MEL 28 Melanoma Human ATCC RPMI 1640 14.0SK-MEL Melanoma Human ATCC RPMI 1640 14.3 28VEGF (expressing VEGF)SK-MEL-1 Melanoma Human ATCC EMEM 1% 14.1 non- essential amino acid 1%Sodium puryvate Panc 96 Pancreatic Human ATCC RPMI 1% 12.5 carcinomaSodium puryvate Panc Pancreatic Human ATCC RPMI 1% 14.2 10.05 carcinomaSodium puryvate Insulin MCF-7 Breast Human ATCC RPMI 1640 9.7 adeno-carcinoma

Example 6 Anticancer Activity In Vitro Against Various Human Tumor CellLines from the U.S. National Cancer Institute Panel

A study measuring the in vitro antitumor activity of ECO-04601 wasperformed by the National Cancer Institute (National Institutes ofHealth, Bethesda, Md., USA) against panel of human cancer cell lines inorder to determine the ECO-04601 concentrations needed to obtain a 50%inhibition of cell proliferation (GI₅₀). The operation of this uniquescreen utilizes 50 different human tumor cell lines, representingleukemia, melanoma and cancers of the lung, colon, brain, ovary, breast,prostate, and kidney.

Culture conditions and plating: The human tumor cell lines of thecancer-screening panel were grown in RPMI 1640 medium containing 5%fetal bovine serum and 2 mM L-glutamine. For a typical screeningexperiment, cells were inoculated into 96 well microtiter plates in 100μL at plating densities ranging from 5,000 to 40,000 cells/welldepending on the doubling time of individual cell lines (Table 5). Aftercell inoculation, the microtiter plates were incubated at 37° C., 5%CO₂, 95% air and 100% relative humidity for 24 h prior to addition ofexperimental drugs. After 24 h, two plates of each cell line were fixedin situ with TCA, to represent a measurement of the cell population foreach cell line at the time of drug addition (Tz).

Evaluation of inhibitory activity on cell proliferation: ECO-04601 wasprovided as a lyophilized powder with an estimated purity of 90+%. Thecompound was stored at −20° C. until day of use. ECO-04601 wassolubilized in dimethyl sulfoxide at 400-fold the desired final maximumtest concentration. At the time of drug addition, an aliquot of frozenconcentrate was thawed and diluted to twice the desired final maximumtest concentration with complete medium containing 50 μg/mL gentamicin.Additional four, 10-fold or ½ log serial dilutions were made to providea total of five drug concentrations plus control. Aliquots of 100 μl ofthese different drug dilutions were added to the appropriate microtiterwells already containing 100 μl of medium, resulting in the requiredfinal drug concentrations (8.0×10⁻⁵ M to 8.0×10⁻⁹ M).

Following drug addition, the plates were incubated for an additional 48h at 37° C., 5% CO₂, 95% air, and 100% relative humidity. For adherentcells, the assay was terminated by the addition of cold TCA. Cells werefixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA(final concentration, 10% TCA) and incubated for 60 minutes at 4° C.Supernatants were discarded, and the plates were washed five times withtap water and air-dried. Sulforhodamine B (SRB) solution (100 μl) at0.4% (w/v) in 1% acetic acid was added to each well, and plates wereincubated for 10 minutes at room temperature. After staining, unbounddye was removed by washing five times with 1% acetic acid and the plateswere air-dried. Bound stain was subsequently solubilized with 10 mMtrizma base, and the absorbance was read on an automated plate reader ata wavelength of 515 nm. For suspension cells, the methodology was thesame except that the assay was terminated by fixing settled cells at thebottom of the wells by gently adding 50 μl of 80% TCA (finalconcentration, 16% TCA).

The growth inhibitory activity of ECO-04601 was measured by NClutilizing the GI₅₀ value, rather than the classical IC₅₀ value. The GI₅₀value emphasizes the correction for the cell count at time zero and,using the seven absorbance measurements [time zero, (Tz), controlgrowth, (C), and test growth in the presence of drug at the fiveconcentration levels (Ti)], GI₅₀ is calculated as[(Ti−Tz)/(C−Tz)]×100=−50, which is the drug concentration resulting in a50% reduction in the net protein increase (as measured by SRB staining)in control cells during the drug incubation.

Result: ECO-04601 shows a significant antitumor activity against severaltypes of tumor as revealed by the NCl screening. Results of the screenare shown in Table 5, and more detailed results of activity againstgliomas are shown in Example 7 (Table 6).

TABLE 5 Inoculation Density (number of Cell Line Name Type Origincells/well) GI₅₀ (×10⁻⁶ M) CCRF-CEM Leukemia Human 40,000 1.08 K-562Leukemia Human 5,000 1.43 RPMI-8226 Leukemia Human 20,000 3.15 A549/ATCCNon-Small Cell Lung Human 7,500 9.10 EKVX Non-Small Cell Lung Human20,000 0.23 HOP-62 Non-Small Cell Lung Human 10,000 8.29 NCl-H226Non-Small Cell Lung Human 20,000 2.00 NCl-H23 Non-Small Cell Lung Human20,000 2.02 NCl-H460 Non-Small Cell Lung Human 7,500 13.60 NCl-H522Non-Small Cell Lung Human 20,000 3.44 COLO 205 Colon Human 15,000 12.70HCT-116 Colon Human 5,000 2.92 HCT-15 Colon Human 10,000 9.73 HT29 ColonHuman 5,000 20.70 SW-620 Colon Human 10,000 2.72 SF-268 CNS Human 15,0004.94 SF-295 CNS Human 10,000 12.70 SF-539 CNS Human 15,000 0.0075 SNB-19CNS Human 15,000 2.90 SNB-75 CNS Human 20,000 7.71 U251 CNS Human 7,5002.19 LOX IMVI Melanoma Human 7,500 4.53 M14 Melanoma Human 15,000 4.57SK-MEL-2 Melanoma Human 20,000 25.0 SK-MEL-28 Melanoma Human 10,000 11.6SK-MEL-5 Melanoma Human 10,000 7.80 UACC-257 Melanoma Human 20,000 2.31UACC-62 Melanoma Human 10,000 1.55 IGR-OV1 Ovarian Human 10,000 3.11OVCAR-3 Ovarian Human 10,000 13.50 OVCAR-4 Ovarian Human 15,000 9.67OVCAR-5 Ovarian Human 20,000 2.81 OVCAR-8 Ovarian Human 10,000 2.65SK-OV-3 Ovarian Human 20,000 4.00 786-0 Renal Human 10,000 6.99 A498Renal Human 25,000 22.30 ACHN Renal Human 10,000 3.10 CAKI-1 Renal Human10,000 15.20 RXF 393 Renal Human 15,000 7.71 SN12C Renal Human 15,0003.85 UO-31 Renal Human 15,000 19.70 DU-145 Prostate Human 10,000 3.56MCF7 Breast Human 10,000 10.10 NCI/ADR-RES Breast Human 15,000 18.30MDA-MB-231/ATCC Breast Human 20,000 2.72 HS 578T Breast Human 20,0002.76 MDA-MB-435 Breast Human 15,000 15.30 BT-549 Breast Human 20,0000.11 T-47D Breast Human 20,000 0.77

The results indicate that ECO-04601 was effective against most of thehuman tumor cell lines that have been assayed in the NCl screening panelsuggesting a broad anticancer activity against several types of humancancer.

Example 7 In Vitro Antiproliferative Study Against a Panel of GliomaCell Lines

The anticancer activity of ECO-04601 was evaluated using a panel ofglioma cancer cell lines shown in Table 6, and the 50% inhibition ofcell proliferation (IC₅₀) was determined.

Culture conditions: The cell lines listed in Table 6 were shown to befree of mycoplasma infection and were maintained on DMEM mediumsupplemented with 10% heat-inactivated fetal bovine serum and 1%penicillin-streptomycin, under 5% CO₂ at 37° C. Cells were passaged oncea week. Prior to use the cells were detached from the culture flask bytreating with trypsin for five to ten minutes. The cells were countedwith a Neubauer glass slide and viability assessed by 0.25% trypan blueexclusion. Only flasks with >95% cell viability, were used in the study.Cell lines amplification and plating: Cells, 5×10³ cells per well in 100μL drug-free medium supplemented with 10% serum, were plated in 96-wellflat bottom microtiter plates and incubated at 37° C. for 48 hrs beforetreatment.Evaluation of inhibitory activity on cell proliferation: Cells (intriplicate wells) were incubated 96 hrs with medium containing differentconcentrations of ECO-04601, starting at 5.0 μg/ml (10 μM). The compoundwas used in a solution of 1% DMSO in D-MEM or RPMI media (or otherequivalent media). The concentrations of ECO-04601 were as follows: 10μM (5.0 μg/ml), 1 μM (0.50 μg/ml), 0.5 μM (0.25 μg/ml), 0.1 μM (0.050μg/ml), 0.5 μM (0.025 μg/ml), 0.01 μM (0.0050 μg/ml), 0.001 μM (0.00050μg/ml). Negative controls were cells treated with vehicle alone (1% DMSOin culture medium). Positive controls were cells treated with 4 to 6increasing concentrations of cisplatin (CDDP) (data not shown). Theoptical density was measured before incubation (time 0) and following 96hrs of incubation with test compound in order to measure the growth rateof each cell line.

At the end of the cell treatment, cell culture media was replaced with150 μl of fresh medium containing 10 mM of4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4. Then50 μl of 2.5 mg/ml of3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide in PBS pH7.4, were added to each well and the culture plates incubated for 4 hrsat 37° C. The resulting supernatant was removed and formazan crystalswere dissolved with 200 μl of DMSO followed by 25 μl of glycine buffer(0.1 M glycine plus 0.1 M NaCl, pH 10.5). The optical density was readin each well using a single wavelength spectrophotometer plate reader at570 nm. Results were expressed as the concentration of drug, whichinhibits 50% of the cell growth (IC₅₀). Each of the cell lines wastested in at least 3 independent experiments.

Results shown in Table 6 confirmed the activity of ECO-04601 againstdifferent brain cancer cell lines including gliosarcoma, which is themost malignant form of type IV glioblastoma multiform. Gliosarcomas area mixture of glial and endothelial cells and are resistant to anychemotherapy.

TABLE 6 Cell IC₅₀ lines Type Origin Source (x 10⁻⁶ M) 9L Gliosarcoma RatATCC 6.82 ± 2.90 GHD Astrocytoma Human ATCC 6.29 ± 2.98 U 373Astrocytoma Human ATCC 3.83 ± 1.37 GL26 Glioblastoma Human ATCC 8.93± 1.10 C6 Glioblastoma Rat ATCC 4.28 ± 2.82 DN Oligodendro- Human ATCC3.26 ± 0.93 glioma GHA Oligodendro- Human ATCC 1.78 ± 0.84 glioma

Example 8 Effect on the Enzymatic Activity of Human Lipoxygenase (5-LO)

5-Lipoxygenase catalyzes the oxidative metabolism of arachidonic acid to5-hydroxyeicosatetraenoic acid (5-HETE), the initial reaction leading toformation of leukotrienes. Eicosanoids derived from arachidonic acid bythe action of lipoxygenases or cycloxygenases have been found to beinvolved in acute and chronic inflammatory diseases (i.e. asthma,multiple sclerosis, rheumatoid arthritis, ischemia, edema) as well inneurodegeneration (Alzheimer disease), aging and various steps ofcarcinogenesis, including tumor promotion, progression and metastasis.

The aim of this study was to determine whether ECO-04601, is able toblock the formation of leukotrienes by inhibiting the enzymatic activityof human 5-LO. Methods employed are based on Carter et al (1991) J.Pharmacol. Exp. Ther. 256(3):929-937, and Safayhi (2000), Planta Medica66:110-113 which are incorporated herein in their entirety by reference.

Experimental Design Human peripheral blood mononuclear cells (PMNs) wereisolated through a Ficoll-Paque density gradient. PMNs were stimulatedby addition A23187 (30 μM final concentration). Stimulated PMNs wereadjusted to a density of 5×10⁶ cells/mL in HBBS medium and incubatedwith the vehicle control (DMSO), ECO-04601 (at final concentrations of0.1, 0.5, 1, 2.5, 5 and 10 μM) and NDGA as positive control (at finalconcentrations of 3, 1, 0.3, 0.1 and 0.03 μM) for 15 minutes at 37° C.Following incubation, samples were neutralized with NaOH andcentrifuged. Leukotriene B4 content was measured in the supernatantusing an Enzyme Immunosorbant Assay (EIA) assay.Results: Results shown in FIG. 8 demonstrated that ECO-04601 inhibitedthe activity of human 5-LO with an apparent IC₅₀=0.93 μM (versus 0.1 μMfor the positive control NDGA) and therefore displays anti-inflammatoryproperties.

Example 9 In Vivo Efficacy in a Glioma Model

The aim of this study was to test whether ECO-04601 administered by i.p.route prevents or delays tumor growth in C6 glioblastoma cell-bearingmice, and to determine an effective dosage regimen.

Animals: A total of 60 six-week-old female mice (Mus musculus nudemice), ranging between 18 to 25 g in weight, were observed for 7 daysbefore treatment. Animal experiments were performed according to ethicalguidelines of animal experimentation (Charte du comité d'éthique duCNRS, juillet 2003) and the English guidelines for the welfare ofanimals in experimental neoplasia (WORKMAN, P., TWENTYMAN, P., BALKWILL,F., et al. (1998). United Kingdom Coordinating Committee on CancerResearch (UKCCCR) Guidelines for the welfare of animals in experimentalneoplasia(Second Edition, July 1997; British Journal of Cancer 77:1-10).Any dead or apparently sick mice were promptly removed and replaced withhealthy mice. Sick mice were euthanized upon removal from the cage.Animals were maintained in rooms under controlled conditions oftemperature (23±2° C.), humidity (45±5%), photoperiodicity (12 hrslight/12 hrs dark) and air exchange. Animals were housed inpolycarbonate cages (5/single cage) that were equipped to provide foodand water. Animal bedding consisted of sterile wood shavings that werereplaced every other day. Food was provided ad libitum, being placed inthe metal lid on the top of the cage. Autoclaved tap water was providedad libitum. Water bottles were equipped with rubber stoppers and sippertubes. Water bottles were cleaned, sterilized and replaced once a week.Two different numbers engraved on two earrings identified the animals.Each cage was labelled with a specific code.Tumor Cell Line: The C6 cell line was cloned from a rat glial tumorinduced by N-nitrosomethyurea (NMU) by Premont et al. (Premont J, BendaP, Jard S., [3H] norepinephrine binding by rat glial cells in culture.Lack of correlation between binding and adenylate cyclase activation.Biochim Biophys Acta. 1975 Feb. 13; 381(2):368-76.) after series ofalternate culture and animal passages.

Cells were grown as adherent monolayers at 37° C. in a humidifiedatmosphere (5% CO₂, 95% air). The culture medium was DMEM supplementedwith 2 mM L-glutamine and 10% fetal bovine serum. For experimental use,tumor cells were detached from the culture flask by a 10 min treatmentwith trypsin-versen. The cells were counted in a hemocytometer and theirviability assessed by 0.25% trypan blue exclusion.

Preparation of the Test Article: for the Test Article, the FollowingProcedure was Followed for reconstitution (performed immediatelypreceding injection). The vehicle consisted of a mixture of benzylalcohol (1.5%), ethanol (8.5%), propylene glycol (27%), PEG 400 (27%),dimethylacetamide (6%) and water (30%). The vehicle solution was firstvortexed in order to obtain a homogeneous liquid. 0.6 mL of the vortexedvehicle solution was added to each vial containing the test article(ECO-04601). Vials were mixed thoroughly by vortexing for 1 minute andinverted and shaken vigorously. Vials were mixed again prior toinjection into each animal.Animal Inoculation with tumor cells: Experiment started at day 0 (D₀).On D₀, mice received a superficial intramuscular injection of C6 tumorcells (5×10⁵ cells) in 0.1 mL of DMEM complete medium into the upperright posterior leg.

Treatment Regimen and Results

In a first series of experiments, treatment started 24 hrs followinginoculation of C6 cells. On the day of the treatment, each mouse wasslowly injected with 100 μL of test or control articles by i.p. route.For all groups, treatment was performed until the tumor volume of thesaline-treated mice (group 1) reached approximately 3 cm³ (around day16). Mice of group 1 were treated daily with a saline isosmotic solutionfor 16 days. Mice of group 2 were treated daily with the vehiclesolution for 16 days. Mice of group 3 were treated daily with 10 mg/kgof ECO-04601 for 16 days. Mice of group 3 were treated every two dayswith 30 mg/kg of ECO-04601 and received 8 treatments. Mice of group 5were treated every three days with 30 mg/kg of ECO-04601 and received 6treatments. Measurement of tumor volume started as soon as tumors becamepalpable (>100 mm³; around day 11 post-inoculation) and was evaluatedevery second day until the end of the treatment using callipers. Asshown in Table 7 and FIG. 9, the mean value of the tumor volume of allECO-04601 treated groups (6 mice/group) was significantly reduced asdemonstrated by the one-way analysis of variance (Anova) test followedby the non-parametric Dunnett's multiple comparison test comparingtreated groups to the saline group. An asterisk in the P value column ofTable 7 indicates a statistically significant value, while “ns”signifies not significant.

TABLE 7 Tumor volume Treatment (mm³) % P Treatment regimen (mean ± SEM)Inhibition value Saline Q1 × 16 3,004.1 ± 249.64 — — Vehicle Q1 × 162,162.0 ± 350.0  28.0% >0.05 ns solution ECO-04601 Q1 × 16 1,220.4 ±283.46 59.4% <0.01* (10 mg/kg) ECO-04601 Q2 × 8  1,236.9 ± 233.99 58.8%<0.01* (30 mg/kg) ECO-04601 Q3 × 6  1,184.1 ± 221.45 60.6% <0.01* (30mg/kg)

In a second series of experiments, treatment started at day 10 followinginoculation of C6 cells when tumors became palpable (around 100 to 200mm³). Treatment was repeated daily for 5 consecutive days. On the day ofthe treatment, each mouse was slowly injected with 100 μL of ECO-04601by i.p. route. Mice of group 1 were treated daily with saline isosmoticsolution. Mice of group 2 were treated daily with the vehicle solution.Mice of group 3 were treated daily with 20 mg/kg of ECO-04601. Mice ofgroup 4 were treated daily with 30 mg/kg of ECO-04601. Mice were treateduntil the tumor volume of the saline-treated control mice (group 1)reached around 4 cm³. Tumor volume was measured every second day untilthe end of the treatment using callipers. As shown in Table 8 and FIG.10, the mean value of the tumor volume of all treated groups (6mice/group) was significantly reduced as demonstrated by the one-wayanalysis of variance (Anova) test followed by the non-parametricDunnett's multiple comparison test comparing treated groups to thesaline group. An asterisk in the P value column of Table 8 indicates astatistically significant value, while “ns” signifies not statisticallysignificant.

Histological analysis of tumor sections showed pronounced morphologicalchanges between ECO-04601-treated tumors and control groups. In tumorstreated with ECO-04601 (20-30 mg/kg), cell density was decreased and thenuclei of remaining tumor cells appeared larger and pycnotic while nosuch changes were observed for vehicle-treated mice (FIG. 11).

TABLE 8 Tumor volume Treatment (mm³) % P Treatment regimen (mean ± SEM)Inhibition value Saline Q1 × 5 4,363.1 ± 614.31 — — Vehicle solution Q1× 5 3,205.0 ± 632.37 26.5% >0.05 ns ECO-04601 Q1 × 5 1,721.5 ± 374.7960.5% <0.01* (20 mg/kg) ECO-04601 Q1 × 5 1,131.6 ± 525.21 74.1% <0.01*(30 mg/kg)

Example 10 Generation of Variants of Eco-04601 According to theInvention

Variants of the ECO-04601 molecule, for example those identified hereinas Formulae III-LIX, can be generated by standard organic chemistryapproaches. General principles of organic chemistry required for makingand manipulating the compounds described herein, including functionalmoieties, reactivity and common protocols are described, for example, in“Advanced Organic Chemistry,” 3^(rd) Edition by Jerry March (1985) whichis incorporated herein by reference in its entirety. In addition, itwill be appreciated by one of ordinary skill in the art that thesynthetic methods described herein may use a variety of protectinggroups, whether or not they are explicitly described. A “protectinggroup” as used herein means a moiety used to block one or morefunctional moieties such as reactive groups including oxygen, sulfur ornitrogen, so that a reaction can be carried out selectively at anotherreactive site in a polyfunctional compound. General principles for theuse of protective groups, their applicability to specific functionalgroups and their uses are described for example in T. H. Greene and P.G. M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) Edition, JohnWiley & Sons, New York (1999).

Scheme 1: Epoxide variants

The epoxide compounds of the present invention (e.g., compoundsaccording to exemplary Formulae VII-XIV) are made from the compound ofFormula II (ECO-04601) by treatment with any of a number of epoxidizingreagents such as perbenzoic acid, monoperphthalic acid or morepreferably by m-chloroperbenzoic acid in an inert solvent such astetrahydrofuran (THF) dichloromethane or 1,2-dichloroethane. It will beappreciated by one of ordinary skill in the art that slightly greaterthan one molecule equivalent of epoxidizing agent will result in themaximal yield of mono-epoxides, and that the reagent, solvent,concentration and temperature of the reaction will dictate the ratio ofspecific mono-epoxides formed. It will also be appreciated that themono-epoxides will be enantiomeric mixtures, and that the di-epoxidesand the tri-epoxide can be prepared as diastereomers and that theconditions of the reaction will determine the ratios of the products.One skilled in the art will appreciate that under most conditions ofreactions the product will be a mixture of all possible epoxides andthat these may be separated by standard methods of chromatography.Exemplary approaches to the generation of mono-, di-, and tri-epoxidesare provided below.

-   -   A) Mono-Epoxides of the Formulae VII, VIII, and IX by        Epoxidation of the Compound of Formula II:

To a solution of the compound of Formula II dissolved in tetrahydrofuran(THF) is added 1.1 equivalents of meta-chloroperbenzoic acid. Thereaction is cooled in an ice bath and stirred at 0° C. for 1-2 hours.The reaction mixture is then evaporated to dryness, re-dissolved inmethanol and subjected to liquid chromatography on a column of SephadexLH-20 to isolate a mixture of predominantly the compounds of FormulaeVII, VIII and IX, contaminated with some unchanged starting material andsome di- and tri-epoxides. The compounds of Formulae VII, VIII and XIXare separated and purified by HPLC using the system described in Example2 for the purification of the compound of Formulae II. In a typicalexperiment yields of 15% to 25% are obtained for each of the compoundsof Formulae VII, VIII and IX.

-   -   B) Synthesis of Compounds of Formulae X, XI, and XII by        Di-Epoxidation of Compound of Formula II:

To a solution of the compound of Formula II dissolved in tetrahydrofuran(THF) is added 2.3 equivalents of meta-chloroperbenzoic acid. Thereaction is cooled in an ice bath and stirred at 0° C. for 1-2 hours.The reaction mixture is then evaporated to dryness, re-dissolved inmethanol and subjected to liquid chromatography on a column of SephadexLH-20 to isolate a mixture of predominantly the compounds of Formulae X,XI and XII, contaminated with traces of unchanged starting material andsome mono- and tri-epoxides. The Compounds of Formulae X, XI and XII areseparated and purified by HPLC using the system described in Example 2for the purification of the compound of Formulae II. In a typicalexperiment, yields of 15% to 20% are obtained for each of the compoundsof Formulae X, XI and XII.

-   -   C) Synthesis of Compound of Formula XIII by Tri-Epoxidation of        Compound of Formula II:

To a solution of the compound of Formula II, dissolved intetrahydrofuran (THF), is added 3.5 equivalents of meta-chloroperbenzoicacid. The reaction is cooled in an ice bath and stirred at 0° C. for 1-2hours. The reaction mixture is then evaporated to dryness, re-dissolvedin methanol and subjected to liquid chromatography on a column ofSephadex LH-20 to isolate the compound of Formula XIII as a mixture ofdiasteriomers in a yield of 80+%.

To a solution of Compound of Formula II dissolved in tetrahydrofuran(THF) is added 1.2 equivalents of acetic anhydride and a few drops oftriethylamine. The reaction mixture allowed to stand at room temperaturefor 1-2 hours and then evaporated to dryness under reduced pressure toobtain the Compound of Formula III in an essentially pure form in analmost quantitative yield

To a solution of Compound of Formula II dissolved in terachloroethyleneis added 1.2 equivalents of the appropriate alkyl bromide (benzylbromide for the compound of formula IV or ethyl bromide for the Compoundof Formula V). The reaction mixture the reaction mixture is heated underreflux for 1-2 hours and then evaporated to dryness under reducedpressure to obtain the Compound of Formula IV or the Compound of FormulaV respectively, in an essentially pure form in an almost quantitativeyield.

A solution of the Compound of Formula II (462 mg) in ethanol (200 ml)with palladium on charcoal (25 mg of 5%) is shaken in an hydrogenationapparatus in an atmosphere of hydrogen. The uptake of hydrogen by thereaction is measured carefully and at the point where one millimole ofhydrogen has been consumed, shaking is stopped, the vessel is rapidlyevacuated and the atmosphere is replaced with nitrogen. The catalyst isremoved by filtration and the filtrate is concentrated to obtain a crudemixture of the Compounds of Formulae XL, XLI and XLII contaminated byunreacted starting material and minor amounts of over reduced products.The desired products may be separated and purified by HPLC or HSCCchromatography using the systems as described in Example 2 above, toobtain approximately 100 mg of each of the Compounds of Formulae XL, XLIand XLII.

A solution of the Compound of Formula II (462 mg) in ethanol (200 ml)with palladium on charcoal (25 mg of 5%) is shaken in an hydrogenationapparatus in an atmosphere of hydrogen. The uptake of hydrogen by thereaction is measured carefully and at the point where two millimoles ofhydrogen has been consumed, shaking is stopped, the vessel is rapidlyevacuated and the atmosphere is replaced with nitrogen. The catalyst isremoved by filtration and the filtrate is concentrated to obtain a crudemixture of the Compounds of Formulae XLIII, XLIV and XLV contaminated bytrace amounts unreacted starting material and minor amounts of under andover reduced products. The desired products may be separated andpurified by HPLC or HSCC chromatography using the systems as describedin Example 2 above, to obtain approximately 100 mg of each of theCompounds of Formulae XLIII, XLIV and XLV.

A solution of the Compound of Formula II (462 mg) in ethanol (200 ml)with palladium on charcoal (25 mg of 5%) is shaken in an hydrogenationapparatus in an atmosphere of hydrogen. The uptake of hydrogen by thereaction is measured carefully and at the point where three millimolesof hydrogen has been consumed, shaking is stopped, the vessel is rapidlyevacuated and the atmosphere is replaced with nitrogen. The catalyst isremoved by filtration and the filtrate is concentrated to obtain anessentially pure sample of the Compound of Formula XLVI

A solution of the Compound of Formula II (100 mg) in acetic anhydride (5ml) is treated with pyridine (250 ul). The reaction mixture is allowedto stand overnight at room temperature and is then diluted with toluene(100 ml). The toluene solution is washed well with aqueous 5% sodiumbicarbonate solutions, then with water and is finally concentrated underreduced pressure to give an essentially pure sample of the Compound ofFormula VI in almost quantitative yield.

A solution of the Compound of Formula VII (100 mg) in tetrahydrofuran(50 ml) is treated with 1N aqueous hydrochloric acid (5 ml). Thereaction mixture is stirred overnight at room temperature and is thendiluted with toluene (100 ml) and water (200 ml). The toluene layer isseparated and the aqueous layer is extracted with a further 100 ml oftoluene. The combined toluene layers are washed once more with water (50ml) and the separated and dried under vacuum to give the vicinal glycolCompound of Formula LI.

A solution of the Compound of Formula II (462 mg) in dry ethyl acetate(200 ml) in an ozonolysis apparatus is cooled to below −20° C. A streamof ozone-containing oxygen is passed into the solution from an ozonegenerator, which has been precalibrated such that the rate of ozonegeneration is known. To obtain predominantly the compound of FormulaXLVII the passage of ozone is halted after 0.9 millimole have beengenerated. To obtain predominantly the compound of Formula XLIX theozone passage is halted after 2 millimoles have been generated and toobtain the compound of Formula LI as the predominant product 3.3millimoles of ozone are generated.

At the completion of the ozonolysis, the reaction mixture is transferredto an hydrogenation apparatus, 5% palladium on calcium carbonatecatalyst (0.2 g) is added to the reaction mixture which is maintained atless than −20° C. and is hydrogenated. When hydrogen uptake is completethe hydrogen atmosphere is replaced with nitrogen and the reactionmixture is allowed to come to room temperature, filtered to removecatalyst and the filtrate is concentrated. The crude product may bepurified by chromatography using either HPLC or HSCC with the systems asdescribed in Example 2 to give, dependent on the amount of ozone used,Compounds of Formulae XLVII, XLIX and LI.

A solution of the Compound of Formula XLVIII (50 mg) in isopropanol (5ml) is cooled in an ice-salt bath and sodium borohydride (10 mg) isadded and the mixture is stirred for 20 minutes. It is then diluted withwater (20 ml) and extracted twice with toluene (10 ml portions) atambient temperature. The combined toluene extracts are filtered and thefiltrate is concentrated to give the Compound of Formula XLVII.

To a solution of Compound of Formula XLII dissolved in tetrahydrofuran(THF) is added 1.1 equivalents of meta-chloroperbenzoic acid. Thereaction is cooled in an ice bath and stirred at 0° C. for 1-2 hours.The reaction mixture is then evaporated to dryness, re-dissolved inmethanol and subjected to liquid chromatography on a column of SephadexLH-20 to isolate a mixture of predominantly the Compounds of FormulaeXIV, and XV, contaminated with some unchanged starting material and somediepoxide. The Compounds of Formulae XIV and XV are separated andpurified by HPLC or HSCC using one of the systems described in Example 2for the purification of the Compound of Formulae II. In a typicalexperiment yields of 35% to 40% are obtained for each of the Compoundsof Formulae XIV and XV.

To a solution of Compound of Formula XL dissolved in tetrahydrofuran(THF) is added 2.2 equivalents of meta-chloroperbenzoic acid. Thereaction is cooled in an ice bath and stirred at 0° C. for 1-2 hours.The reaction mixture is then evaporated to dryness, re-dissolved inmethanol and subjected to liquid chromatography on a column of SephadexLH-20 to isolate essentially pure Compound of Formulae XIX in goodyield.

To a solution of Compound of Formula II dissolved in toluene (9 parts)tetrahydrofuran (1 part), cooled in an ice-bath is added 1.1 equivalentsof acetic anhydride and two drops of boron trifluoride etherate. Thereaction is maintained cool in an ice bath and stirred at 0° C. for 1-2hours. The reaction mixture is then poured into aqueous 5% sodiumbicarbonate solution shaken and the toluene layer is removed. Theaqueous layer is re-extracted with toluene and the combined toluenelayers are concentrated to a mixture of predominantly the Compounds ofFormulae XXVI, XXVII and XXVIII, contaminated with some unchangedstarting material and some diacetates. The Compounds of Formulae XXVI,XXVII and XXVIII are separated and purified by HPLC or HSCC using one ofthe systems described in Example 2 for the purification of the Compoundof Formulae II. In a typical experiment yields of 25% to 30% areobtained for each of the Compounds of Formulae XXVI, XXVII and XXVIII.

A solution of the Compound of Formula II (1 g) in tetrahydrofuran 50(ml) is titrated with exactly one equivalent of sodium methoxide,allowed to stand for 30 minutes at room temperature and then treatedwith 1.2 equivalents of dimethylsulphate. Heat the mixture under refluxfor one hour, cool to room temperature and pour into a mixture oftoluene (200 ml) and water (200 ml). The layers are separated and theaqueous layer is extracted once more with an equal portion of toluene.The combined toluene layers are washed once with 1N aqueous acetic acidand then concentrated to s crude product, which is predominantly amixture of the Compounds of Formulae XXXIII, XXXIV and XXXV with someunchanged starting material and traces of over-methylated derivatives.The desired products may be separated and purified by HPLC or HSCCchromatography using the systems as described in Example 2 above, toobtain approximately 200 mg of each of the Compounds of Formulae XXXIII,XXXIV and XXXV.

Example 11 Genes and Proteins for the Production of Compounds of Formula

Micromonospora sp. strain 046-ECO11 is a representative microorganismuseful in the production of the compound of the invention. Strain046-ECO11 has been deposited with the International Depositary Authorityof Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 ArlingtonStreet, Winnipeg, Manitoba, Canada R3E 3R2 on Mar. 7, 2003 and wasassigned IDAC accession no. 070303-01. The biosynthetic locus for theproduction of the compound of Formula II was identified in the genome ofMicromonospora sp. strain 046-ECO11 using the genome scanning methoddescribed in U.S. Ser. No. 10/232,370, CA 2,352,451 and Zazopoulos et.al., Nature Biotechnol., 21, 187-190 (2003).

The biosynthetic locus spans approximately 52,400 base pairs of DNA andencodes 43 proteins. More than 10 kilobases of DNA sequence wereanalyzed on each side of the locus and these regions were deemed tocontain primary genes or genes unrelated to the synthesis of thecompound of Formula II. As illustrated in FIG. 12, the locus iscontained within three sequences of contiguous base pairs, namely Contig1 having the 36,602 contiguous base pairs of SEQ ID NO: 1 and comprisingORFs 1 to 31 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61and 63), Contig 2 having the 5,960 contiguous base pairs of SEQ ID NO:64 and comprising ORFs 32 to 35 (SEQ ID NOS: 66, 68, 70 and 72), andContig 3 having the 9,762 base pairs of SEQ ID NO: 73 and comprisingORFs 36 to 43 (SEQ ID NOS: 75, 77, 79, 81, 83, 85, 87 and 89). Theorder, relative position and orientation of the 43 open reading framesrepresenting the proteins of the biosynthetic locus are illustratedschematically in FIG. 12. The top line in FIG. 12 provides a scale inbase pairs. The gray bars depict the three DNA contigs (SEQ ID NOS: 1,64 and 73) that cover the locus. The empty arrows represent the 43 openreading frames of this biosynthetic locus. The black arrows representthe two deposited cosmid clones covering the locus.

The biosynthetic locus will be further understood with reference to thesequence listing which provides contiguous nucleotide sequences anddeduced amino acid sequences of the locus from Micromonospora sp. strain046-ECO11. The contiguous nucleotide sequences are arranged such that,as found within the biosynthetic locus, Contig 1 (SEQ ID NO: 1) isadjacent to the 5′ end of Contig 2 (SEQ ID NO: 64), which in turn isadjacent to Contig 3 (SEQ ID NO: 73). The ORFs illustrated in FIG. 12and provided in the sequence listing represent open reading framesdeduced from the nucleotide sequences of Contigs 1, 2 and 3 (SEQ ID NOS:1, 64 and 73). Referring to the Sequence Listing, ORF 1 (SEQ ID NO: 3)is the polynucleotide drawn from residues 2139 to 424 of SEQ ID NO: 1,and SEQ ID NO: 2 represents that polypeptide deduced from SEQ ID NO: 3.ORF 2 (SEQ ID NO: 5) is the polynucleotide drawn from residues 2890 to4959 of SEQ ID NO: 1, and SEQ ID NO: 4 represents the polypeptidededuced from SEQ ID NO: 5. ORF 3 (SEQ ID NO: 7) is the polynucleotidedrawn from residues 7701 to 5014 of SEQ ID NO: 1, and SEQ ID NO: 6represents the polypeptide deduced from SEQ ID NO: 7. ORF 4 (SEQ ID NO:9) is the polynucleotide drawn from residues 8104 to 9192 of SEQ ID NO:1, and SEQ ID NO: 8 represents the polypeptide deduced from SEQ ID NO:9. ORF 5 (SEQ ID NO: 11) is the polynucleotide drawn from residues 9192to 10256 of SEQ ID NO: 1, and SEQ ID NO: 10 represents the polypeptidededuced from SEQ ID NO: 11. ORF 6 (SEQ ID NO: 13) is the polynucleotidedrawn from residues 10246 to 11286 of SEQ ID NO: 1, and SEQ ID NO: 12represents the polypeptide deduced from SEQ ID NO: 13. ORF 7 (SEQ ID NO:15) is the polynucleotide drawn from residues 11283 to 12392 of SEQ IDNO: 1, and SEQ ID NO: 14 represents the polypeptide deduced from SEQ IDNO: 15. ORF 8 (SEQ ID NO: 17) is the polynucleotide drawn from residues12389 to 13471 of SEQ ID NO: 1, and SEQ ID NO: 16 represents thepolypeptide deduced from SEQ ID NO: 17. ORF 9 (SEQ ID NO: 19) is thepolynucleotide drawn from residues 13468 to 14523 of SEQ ID NO: 1, andSEQ ID NO: 18 represents the polypeptide deduced from SEQ ID NO: 19. ORF10 (SEQ ID NO: 21) is the polynucleotide drawn from residues 14526 to15701 of SEQ ID NO: 1, and SEQ ID NO: 20 represents the polypeptidededuced from SEQ ID NO: 21. ORF 11 (SEQ ID NO: 23) is the polynucleotidedrawn from residues 15770 to 16642 of SEQ ID NO: 1, and SEQ ID NO: 22represents the polypeptide deduced from SEQ ID NO: 23. ORF 12 (SEQ IDNO: 25) is the polynucleotide drawn from residues 16756 to 17868 of SEQID NO: 1, and SEQ ID NO: 24 represents the polypeptide deduced from SEQID NO: 25. ORF 13 (SEQ ID NO: 27) is the polynucleotide drawn fromresidues 17865 to 18527 of SEQ ID NO: 1, and SEQ ID NO: 26 representsthe polypeptide deduced from SEQ ID NO: 27. ORF 14 (SEQ ID NO: 29) isthe polynucleotide drawn from residues 18724 to 19119 of SEQ ID NO: 1,and SEQ ID NO: 28 represents the polypeptide deduced from SEQ ID NO: 29.ORF 15 (SEQ ID NO: 31) is the polynucleotide drawn from residues 19175to 19639 of SEQ ID NO: 1, and SEQ ID NO: 30 represents the polypeptidededuced from SEQ ID NO: 31. ORF 16 (SEQ ID NO: 33) is the polynucleotidedrawn from residues 19636 to 21621 of SEQ ID NO: 1, and SEQ ID NO: 32represents the polypeptide deduced from SEQ ID NO: 33. ORF 17 (SEQ IDNO: 35) is the polynucleotide drawn from residues 21632 to 22021 of SEQID NO: 1, and SEQ ID NO: 34 represents the polypeptide deduced from SEQID NO: 35. ORF 18 (SEQ ID NO: 37) is the polynucleotide drawn fromresidues 22658 to 22122 of SEQ ID NO: 1, and SEQ ID NO: 36 representsthe polypeptide deduced from SEQ ID NO: 37. ORF 19 (SEQ ID NO: 39) isthe polynucleotide drawn from residues 24665 to 22680 of SEQ ID NO: 1,and SEQ ID NO: 38 represents the polypeptide deduced from SEQ ID NO: 39.ORF 20 (SEQ ID NO: 41) is the polynucleotide drawn from residues 24880to 26163 of SEQ ID NO: 1, and SEQ ID NO: 40 represents the polypeptidededuced from SEQ ID NO: 41. ORF 21 (SEQ ID NO: 43) is the polynucleotidedrawn from residues 26179 to 27003 of SEQ ID NO: 1, and SEQ ID NO: 42represents the polypeptide deduced from SEQ ID NO: 43. ORF 22 (SEQ IDNO: 45) is the polynucleotide drawn from residues 27035 to 28138 of SEQID NO: 1, and SEQ ID NO: 44 represents the polypeptide deduced from SEQID NO: 45. ORF 23 (SEQ ID NO: 47) is the polynucleotide drawn fromresidues 28164 to 28925 of SEQ ID NO: 1, and SEQ ID NO: 46 representsthe polypeptide deduced from SEQ ID NO: 47. ORF 24 (SEQ ID NO: 49) isthe polynucleotide drawn from residues 28922 to 30238 of SEQ ID NO: 1,and SEQ ID NO: 48 represents the polypeptide deduced from SEQ ID NO: 49.ORF 25 (SEQ ID NO: 51) is the polynucleotide drawn from residues 30249to 31439 of SEQ ID NO: 1, and SEQ ID NO: 50 represents the polypeptidededuced from SEQ ID NO: 51. ORF 26 (SEQ ID NO: 53) is the polynucleotidedrawn from residues 31439 to 32224 of SEQ ID NO: 1, and SEQ ID NO: 52represents the polypeptide deduced from SEQ ID NO: 53. ORF 27 (SEQ IDNO: 55) is the polynucleotide drawn from residues 32257 to 32931 of SEQID NO: 1, and SEQ ID NO: 54 represents the polypeptide deduced from SEQID NO: 55. ORF 28 (SEQ ID NO: 57) is the polynucleotide drawn fromresidues 32943 to 33644 of SEQ ID NO: 1, and SEQ ID NO: 56 representsthe polypeptide deduced from SEQ ID NO: 57. ORF 29 (SEQ ID NO: 59) isthe polynucleotide drawn from residues 34377 to 33637 of SEQ ID NO: 1,and SEQ ID NO: 58 represents the polypeptide deduced from SEQ ID NO: 59.ORF 30 (SEQ ID NO: 61) is the polynucleotide drawn from residues 34572to 34907 of SEQ ID NO: 1, and SEQ ID NO: 60 represents the polypeptidededuced from SEQ ID NO: 61. ORF 31 (SEQ ID NO: 63) is the polynucleotidedrawn from residues 34904 to 36583 of SEQ ID NO: 1, and SEQ ID NO: 62represents the polypeptide deduced from SEQ ID NO: 63. ORF 32 (SEQ IDNO: 66) is the polynucleotide drawn from residues 23 to 1621 of SEQ IDNO: 64, and SEQ ID NO: 65 represents the polypeptide deduced from SEQ IDNO: 66. ORF 33 (SEQ ID NO: 68) is the polynucleotide drawn from residues1702 to 2973 of SEQ ID NO: 64, and SEQ ID NO: 67 represents thepolypeptide deduced from SEQ ID NO: 68. ORF 34 (SEQ ID NO: 70) is thepolynucleotide drawn from residues 3248 to 4270 of SEQ ID NO: 64, andSEQ ID NO: 69 represents the polypeptide deduced from SEQ ID NO: 70. ORF35 (SEQ ID NO: 72) is the polynucleotide drawn from residues 4452 to5933 of SEQ ID NO: 64, and SEQ ID NO: 71 represents the polypeptidededuced from SEQ ID NO: 72. ORF 36 (SEQ ID NO: 75) is the polynucleotidedrawn from residues 30 to 398 of SEQ ID NO: 73, and SEQ ID NO: 74represents the polypeptide deduced from SEQ ID NO: 75. ORF 37 (SEQ IDNO: 77) is the polynucleotide drawn from residues 395 to 1372 of SEQ IDNO: 73, and SEQ ID NO: 76 represents the polypeptide deduced from SEQ IDNO: 77. ORF 38 (SEQ ID NO: 79) is the polynucleotide drawn from residues3388 to 1397 of SEQ ID NO: 73, and SEQ ID NO: 78 represents thepolypeptide deduced from SEQ ID NO: 79. ORF 39 (SEQ ID NO: 81) is thepolynucleotide drawn from residues 3565 to 5286 of SEQ ID NO: 73, andSEQ ID NO: 80 represents the polypeptide deduced from SEQ ID NO: 81. ORF40 (SEQ ID NO: 83) is the polynucleotide drawn from residues 5283 to7073 of SEQ ID NO: 73, and SEQ ID NO: 82 represents the polypeptidededuced from SEQ ID NO: 83. ORF 41 (SEQ ID NO: 85) is the polynucleotidedrawn from residues 7108 to 8631 of SEQ ID NO: 73, and SEQ ID NO: 84represents the polypeptide deduced from SEQ ID NO: 85. ORF 42 (SEQ IDNO: 87) is the polynucleotide drawn from residues 9371 to 8673 of SEQ IDNO: 73, and SEQ ID NO: 86 represents the polypeptide deduced from SEQ IDNO: 87. ORF 43 (SEQ ID NO: 89) is the polynucleotide drawn from residues9762 to 9364 of SEQ ID NO: 73, and SEQ ID NO: 88 represents thepolypeptide deduced from SEQ ID NO: 89.

Some open reading frames provided in the Sequence Listing, namely ORF 2(SEQ ID NO: 5), ORF 5 (SEQ ID NO: 11), ORF 12 (SEQ ID NO: 25), ORF 13(SEQ ID NO: 27), ORF 15 (SEQ ID NO: 31), ORF 17 (SEQ ID NO: 35), ORF 19(SEQ ID NO: 39), ORF 20 (SEQ ID NO: 41), ORF 22 (SEQ ID NO: 45), ORF 24(SEQ ID NO: 49), ORF 26 (SEQ ID NO: 53) and ORF 27 (SEQ ID NO: 55)initiate with non-standard initiation codons (eg. GTG-Valine, orCTG-Leucine) rather than standard initiation codon ATG methionine. AllORFs are listed with the appropriate M, V or L amino acids at theamino-terminal position to indicate the specificity of the first codonof the ORF. It is expected, however, that in all cases thebiosynthesized protein will contain a methionine residue, and morespecifically a formylmethionine residue, at the amino terminal position,in keeping with the widely accepted principle that protein synthesis inbacteria initiate with methionine (formylmethionine) even when theencoding gene specifies a non-standard initiation codon (e.g. StryerBioChemistry 3^(rd) edition, 1998, W.H. Freeman and Co., New York, pp.752-754).

ORF 32 (SEQ ID NO: 65) is incomplete and contains a truncation of 10 to20 amino acids from its carboxy terminus. This is due to incompletesequence information between Contigs 2 and 3 (SEQ ID NOS: 64 and 73,respectively).

Deposits of E. coli DH10B vectors, each harbouring a cosmid clone(designated in FIG. 12 as 046KM and 046KQ respectively) of a partialbiosynthetic locus for the compound of Formula II from Micromonosporasp. strain 046-ECO11 and together spanning the full biosynthetic locusfor production of the compound of Formula II have been deposited withthe International Depositary Authority of Canada, Bureau ofMicrobiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba,Canada R3E 3R2 on Feb. 25, 2003. The cosmid clone designated 046KM wasassigned deposit accession numbers IDAC 250203-06, and the cosmid clonedesignated 046KQ was assigned deposit accession numbers IDAC 250203-07.Cosmid 046KM covers residue 1 to residue 32,250 of Contig 1 (SEQ ID NO:1). Cosmid 046KQ covers residue 21,700 of Contig 1 (SEQ ID NO: 1) toresidue 9,762 of Contig 3 (SEQ ID NO: 73). The sequence of thepolynucleotides comprised in the deposited strains, as well as the aminoacid sequence of any polypeptide encoded thereby are controlling in theevent of any conflict with any description of sequences herein.

The deposit of the deposited strains has been made under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The deposited strainswill be irrevocably and without restriction or condition released to thepublic upon the issuance of a patent. The deposited strains are providedmerely as convenience to those skilled in the art and are not anadmission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A license may be required to make, use orsell the deposited strains, and compounds derived therefrom, and no suchlicense is hereby granted.

In order to identify the function of the proteins coded by the genesforming the biosynthetic locus for the production of the compound ofFormula II the gene products of ORFs 1 to 43, namely SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78,80, 82, 84, 86 and 88 were compared, using the BLASTP version 2.2.10algorithm with the default parameters, to sequences in the NationalCenter for Biotechnology Information (NCBI) nonredundant proteindatabase and the DECIPHER® database of microbial genes, pathways andnatural products (Ecopia BioSciences Inc. St.-Laurent, QC, Canada).

The accession numbers of the top GenBank™ hits of this BLAST analysisare presented in Table 14 along with the corresponding E values. The Evalue relates the expected number of chance alignments with an alignmentscore at least equal to the observed alignment score. An E value of 0.00indicates a perfect homolog. The E values are calculated as described inAltschul et al. J. Mol. Biol., 215, 403-410 (1990). The E value assistsin the determination of whether two sequences display sufficientsimilarity to justify an inference of homology.

TABLE 14 ORF Family # aa GenBank homology Probability % Identity %Similarity Proposed function of GenBank match 1 ABCC 571 NP_736627.1,590aa 1.00E−107 222/496 (44.76%) 278/496 (56.05%) ABC transporterCorynebacterium efficiens NP_600638.1, 510aa 5.00E−80 184/500 (36.8%)260/500 (52%) ABC transporter Corynebacterium efficiens NP_600638.1,510aa 3.00E−12  58/195 (29.74%)  84/195 (43.08%) ABC transporterCorynebacterium efficiens 2 RECH 689 CAC93719.1, 923aa 3.00E−17  57/158(36.08%)  87/158 (55.06%) regulator[Lechevalieria aerocolonigenes]BAC55205.1, 943aa 3.00E−12  51/170 (30%)  81/170 (47.65%)transcriptional activator [Streptomyces sp. NP_631154.1, 932aa 3.00E−07 29/63 (46.03%)  40/63 (63.49%) regulator. [Streptomyces coelicolorA3(2) 3 REGD 895 CAC93719.1, 923aa 3.00E−20  92/330 (27.88%) 142/330(43.03%) regulator [Lechevalieria aerocolonigenes] BAC55205.1, 943aa1.00E−15  80/277 (28.88%) 101/277 (36.46%) activator [Streptomyces sp.TP-A0274] NP_733725.1, 908aa 3.00E−12  95/339 (28.02%) 140/339 (41.3%)regulator [Streptomyces coelicolor A3(2)] 4 IDSA 362 NP_601376.2, 371aa2.00E−80 158/321 (49.22%) 208/321 (64.8%) GGPP synthase [Corynebacteriumglutamicum NP_738677.1, 366aa 3.00E−79 158/330 (47.88%) 204/330 (61.82%)polyprenyl synthase, Corynebacterium efficiens NP_216689.1, 352aa2.00E−78 153/331 (46.22%) 203/331 (61.33%) idsA2 [Mycobacteriumtuberculosis H37Rv] 5 MVKA 354 BAB07790.1, 345aa 2.00E−71 150/326(46.01%) 193/326 (59.2%) mevalonate kinase [Streptomyces sp. CL190]BAB07817.1, 334aa 5.00E−66 145/324 (44.75%) 185/324 (57.1%) mevalonatekinase [Kitasatospora griseola] NP_720650.1, 332aa 3.00E−36  95/327(29.05%) 157/327 (48.01%) mevalonate kinase [Streptococcus mutans 6 DMDA346 BAB07791.1, 350aa 2.00E−88 177/305 (58.03%) 199/305 (65.25%)diphosphomevalonate decarboxylase [Streptomyces sp. BAB07818.1, 300aa2.00E−69 145/275 (52.73%) 168/275 (61.09%) mevalonate diPHdecaroboxylase [Kitasatospora griseola] NP_785307.1, 325aa 3.00E−44105/307 (34.2%) 141/307 (45.93%) diphosphomevalonate decarboxylase[Lactobacillus plantarum 7 MVKP 369 BAB07792.1, 374aa 4.00E−93 183/365(50.14%) 220/365 (60.27%) phosphomevalonate kinase [Streptomyces sp.CL190] BAB07819.1, 360aa 6.00E−77 171/358 (47.77%) 202/358 (56.42%)phosphomevalonate kinase [Kitasatospora griseola] AAG02442.1, 368aa2.00E−31 102/354 (28.81%) 149/354 (42.09%) 3 phosphomevalonate kinase[Enterococcus faecalis] 8 IPPI 360 Q9KWF6, 364aa 1.00E−128 238/361(65.93%) 269/361 (74.52%) Isopentenyl-diphosphate delta-isomeraseQ9KWG2, 363aa 1.00E−128 230/349 (65.9%) 270/349 (77.36%)Isopentenyl-diphosphate delta-isomerase NP_814639.1, 347aa 5.00E−73154/348 (44.25%) 212/348 (60.92%) isopentenyl diphosphate isomerase[Enterococcus faecalis 9 HMGA 351 BAA70975.1, 353aa 1.00E−165 284/348(81.61%) 317/348 (91.09%) 3-hydroxy-3-methylglutaryl coenzyme Areductase [Streptomyces sp.] BAA74565.1, 353aa 1.00E−160 282/347(81.27%) 310/347 (89.34%) 3-hydroxy-3-methylglutaryl coenzyme Areductase [Kitasatospora griseola] BAA74566.1, 353aa 1.00E−155 277/347(79.83%) 299/347 (86.17%) 3-hydroxy-3-methylglutaryl coenzyme Areductase [Streptomyces sp.] 10 KASH 391 BAB07795.1, 389aa 1.00E−148260/386 (67.36%) 300/386 (77.72%) 3-hydroxy-3-methylglutaryl CoAsynthase [Streptomyces sp. CL190] BAB07822.1, 346aa 1.00E−136 239/343(69.68%) 268/343 (78.13%) HMG-CoA synthase [Kitasatospora griseola]CAD24420.1, 388aa 6.00E−79 166/385 (43.12%) 210/385 (54.55%) HMG-CoAsynthase [Paracoccus zeaxanthinifaciens] 11 IPTN 290 NP_631248.1, 295aa5.00E−22  79/282 (28.01%) 124/282 (43.97%) hypothetical protein[Streptomyces coelicolor A3(2)] AAN65239.1, 324aa 5.00E−06  70/278(25.18%) 112/278 (40.29%) cloQ [Streptomyces roseochromogenes subsp.oscitans] 12 SPKG 370 AAM78435.1, 344aa 5.00E−48 112/208 (53.85%)131/208 (62.98%) two-component sensor [Streptomyces coelicolor A3(2)]NP_630507.1, 382aa 5.00E−48 112/208 (53.85%) 131/208 (62.98%) sensorkinase [Streptomyces coelicolor A3(2)] ZP_00058991.1, 407aa 9.00E−34 88/198 (44.44%) 114/198 (57.58%) Signal transduction histidine kinase[Thermobifida fusca] 13 RREB 220 NP_630508.1, 224aa 3.00E−79 148/220(67.27%) 179/220 (81.36%) regulatory protein [Streptomyces coelicolorA3(2)] ZP_00058992.1, 221aa 4.00E−67 129/218 (59.17%) 163/218 (74.77%)Response regulator [Thermobifida fusca] NP_625364.1, 221aa 6.00E−66134/222 (60.36%) 164/222 (73.87%) response regulator [Streptomycescoelicolor A3(2)] 14 UNES 131 No hit — — — — 15 UNEZ 154 NP_649459.2,628aa 7.60E−02  21/55 (38.18%)  33/55 (60%) CG1090-PB [Drosophilamelanogaster] NP_730819.1, 473aa 7.60E−02  21/55 (38.18%)  33/55 (60%)CG1090-PA [Drosophila melanogaster] AAM11079.1, 428aa 7.60E−02  21/55(38.18%)  33/55 (60%) GH23040p [Drosophila melanogaster] 16 OXDS 661NP_242948.1, 500aa 1.00E−52 129/433 (29.79%) 197/433 (45.5%) unknownconserved protein [Bacillus halodurans] ZP_00091617.1, 480aa 3.00E−32123/426 (28.87%) 175/426 (41.08%) Putative multicopper oxidases[Azotobacter vinelandii] NP_252457.1, 463aa 1.00E−31 115/408 (28.19%)170/408 (41.67%) metallo-oxidoreductase [Pseudomonas aeruginosa PA01] 17UNFD 129 NP_437360.1, 127aa 7.00E−33  73/121 (60.33%)  87/121 (71.9%)bleomycin resistance protein family [Sinorhizobium meliloti] AAO91879.1,123aa 1.00E−31  68/117 (58.12%)  86/117 (73.5%) unknown [unculturedbacterium] NP_103287.1, 131aa 1.00E−23  59/122 (48.36%)  76/122 (62.3%)unknown protein [Mesorhizobium loti] 18 UNFA 178 19 CSMB 661ZP_00137697.1, 769aa 1.00E−166 319/622 (51.29%) 408/622 (65.59%)Anthranilate/para-aminobenzoate synthase [Pseudomonas aeruginosaNP_250594.1, 627aa 1.00E−166 319/622 (51.29%) 408/622 (65.59%) phenazinebiosynthesis protein PhzE [Pseudomonas aeruginosa PA01] ZP_00137701.1,687aa 1.00E−166 319/622 (51.29%) 408/622 (65.59%)Anthranilate/para-aminobenzoate synthas [Pseudomonas aeruginosa 20 AAKD427 P41403, 421aa 1.00E−64 161/420 (38.33%) 214/420 (50.95%)Aspartokinase (Aspartate kinase) ZP_00057166.1, 445aa 2.00E−64 154/415(37.11%) 218/415 (52.53%) Aspartokinases [Thermobifida fusca]AAD49567.1, 421aa 6.00E−64 152/412 (36.89%) 216/412 (52.43%)aspartokinase subunit A [Amycolatopsis mediterranei] 21 ALDB 274NP_275722.1, 266aa 2.00E−53 104/231 (45.02%) 147/231 (63.64%) conservedprotein [Methanothermobacter thermautotrophicus] NP_614692.1, 270aa2.00E−52 104/240 (43.33%) 146/240 (60.83%) Fructose-1,6-bisphosphatealdolase [Methanopyrus kandleri AV19] NP_615406.1, 267aa 2.00E−50 99/231 (42.86%) 141/231 (61.04%) fructose-bisphosphate aldolase[Methanosarcina acetivorans str. C2A] 22 UNFC 367 NP_275723.1, 378aa4.00E−46 116/308 (37.66%) 171/308 (55.52%) conserved protein[Methanothermobacter thermautotrophicus] NP_614691.1, 402aa 2.00E−45115/295 (38.98%) 163/295 (55.25%) alternative 3-dehydroquinate synthase[Methanopyrus kandleri NP_248244.1, 361aa 2.00E−43 103/255 (40.39%)150/255 (58.82%) conserved hypothetical protein [Methanococcusjannaschii 23 HYDK 253 NP_577771.1, 247aa 4.00E−14  55/178 (30.9%) 87/178 (48.88%) metal-dependent hydrolase [Pyrococcus furiosus DSM3638] NP_142108.1, 247aa 1.00E−12  50/151 (33.11%)  78/151 (51.66%)hypothetical protein PH0093 [Pyrococcus horikoshii] NP_125791.1, 248aa1.00E−11  42/151 (27.81%)  76/151 (50.33%) hypothetical protein[Pyrococcus abyssi] 24 ADSA 438 NP_070499.1, 433aa 2.00E−41 122/347(35.16%) 171/347 (49.28%) coenzyme F390 synthetase [Archaeoglobusfulgidus NP_618724.1, 434aa 5.00E−41 119/345 (34.49%) 171/345 (49.57%)coenzyme F390 synthetase [Methanosarcina acetivorans NP_632700.1, 437aa7.00E−41 121/345 (35.07%) 171/345 (49.57%) Coenzyme F390 synthetase[Methanosarcina mazei Goe1] 25 HOXV 396 ZP_00027430.1, 442aa 8.00E−76152/358 (42.46%) 211/358 (58.94%) 2-polyprenyl-6-methoxyphenolhydroxylase [Burkholderia fungorum] NP_627457.1, 420aa 1.00E−71 161/420(38.33%) 216/420 (51.43%) salicylate hydroxylase [Streptomycescoelicolor A3(2)] ZP_00033877.1, 403aa 2.00E−68 146/395 (36.96%) 200/395(50.63%) 2-polyprenyl-6-methoxyphenol hydroxylase [Burkholderiafungorum] 26 SDRA 261 NP_391080.1, 261aa 6.00E−58 119/261 (45.59%)149/261 (57.09%) 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase[Bacillus subtilis] ZP_00059512.1, 260aa 1.00E−55 116/259 (44.79%)144/259 (55.6%) Dehydrogenase [Thermobifida fusca] AAG31126.1, 257aa9.00E−55 117/257 (45.53%) 144/257 (56.03%) MxcC [Stigmatella aurantiaca]27 DHBS 224 Q51790, 207aa 7.00E−60 110/198 (55.56%) 142/198 (71.72%)isochorismatase Q51518, 207aa 1.00E−58 110/198 (55.56%) 140/198 (70.71%)isochorismatase NP_391077.1, 312aa 2.00E−58 106/203 (52.22%) 139/203(68.47%) isochorismatase [Bacillus subtilis] 28 SDRA 233 NP_103491.1,242aa 9.00E−21  74/230 (32.17%) 112/230 (48.7%) acyl-carrier proteinreductase [Mesorhizobium loti] AAL14912.1, 245aa 1.00E−15  65/229(28.38%) 100/229 (43.67%) short-chain dehydrogenase [Rhizobiumleguminosarum bv. trifolii] NP_902480.1, 235aa 7.00E−15  67/229 (29.26%)100/229 (43.67%) oxidoreductase [Chromobacterium violaceum 29 UNIQ 246S18541, 281aa 4.50E−02  43/146 (29.45%)  63/146 (43.15%) hypotheticalprotein 3 - Streptomyces coelicolor NP_629228.1, 281aa 5.90E−02  43/146(29.45%)  63/146 (43.15%) hypothetical protein [Streptomyces coelicolorA3(2)] 30 UNFE 111 ZP_00058149.1, 130aa 1.00E−10  35/97 (36.08%)  47/97(48.45%) membrane protein [Thermobifida fusca] NP_737701.1, 120aa1.00E−09  37/111 (33.33%)  51/111 (45.95%) hypothetical protein[Corynebacterium efficiens NP_827629.1, 118aa 7.00E−09  35/105 (33.33%) 51/105 (48.57%) hypothetical protein [Streptomyces avermitilis MA-4680]31 EFFT 559 ZP_00058148.1, 537aa 2.00E−67 165/517 (31.91%) 253/517(48.94%) Predicted symporter [Thermobifida fusca] NP_626090.1, 544aa4.00E−66 162/521 (31.09%) 257/521 (49.33%) transport protein[Streptomyces coelicolor A3(2)] NP_827630.1, 549aa 7.00E−63 160/523(30.59%) 256/523 (48.95%) sodium-dependent symporter [Streptomycesavermitilis 32 HOYH 532 AAM96655.1, 544aa 2.00E−92 206/526 (39.16%)279/526 (53.04%) 2,4-dihydroxybenzoate monooxygenase [Sphingobiumchlorophenolicum] ZP_00029353.1, 543aa 1.00E−73 188/539 (34.88%) 263/539(48.79%) 2-polyprenyl-6-methoxyphenol hydroxylase [Burkholderiafungorum] NP_769326.1, 569aa   5e−62 173/519 (33.33%) 251/519 (48.36%)blr2686 [Bradyrhizobium japonicum] dbj 33 DAHP 423 T03226, 391aa1.00E−111 207/383 (54.05%) 259/383 (67.62%) hypothetical protein -Streptomyces hygroscopicus ZP_00137693.1, 405aa 3.00E−87 172/385(44.68%) 233/385 (60.52%) DAHP synthase [Pseudomonas aeruginosaUCBPP-PA14] NP_250592.1, 405aa 1.00E−86 169/380 (44.47%) 232/380(61.05%) phenazine biosynthesis protein PhzC [Pseudomonas aeruginosa 34REGG 340 BAC53615.1, 346aa 1.00E−67 142/307 (46.25%) 192/307 (62.54%)regulator protein [Streptomyces kasugaensis] S44506, 424aa 3.00E−66141/305 (46.23%) 182/305 (59.67%) regulator protein - Streptomycesglaucescens AAK81822.1, 348aa 1.00E−65 141/323 (43.65%) 192/323 (59.44%)transcriptional regulator [Streptomyces lavendulae] 35 UNFJ 493ZP_00073237.1, 678aa 7.00E−35 124/454 (27.31%) 197/454 (43.39%) RTXtoxins [Trichodesmium erythraeum IMS101] NP_484716.1, 433aa 3.00E−05109/470 (23.19%) 172/470 (36.6%) similar to vanadium chloroperoxidase[Nostoc sp. ZP_00067005.1, 667aa 7.40E−02  37/139 (26.62%)  52/139(37.41%) hypothetical protein [Microbulbifer degradans 2-40] 36 RECI 112NP_627088.1, 125aa 3.00E−17  48/100 (48%)  59/100 (59%) hypotheticalprotein [Streptomyces coelicolor A3(2)] NP_846017.1, 109aa 7.00E−15 40/101 (39.6%)  60/101 (59.41%) hypothetical protein [Bacillusanthracis str. Ames] NP_241272.1, 174aa 9.00E−15  39/106 (36.79%) 62/106 (58.49%) unknown conserved protein [Bacillus halodurans] 37 UNIQ325 NP_422203.1, 187aa 1.00E−03  24/61 (39.34%)  36/61 (59.02%)hypothetical protein [Caulobacter crescentus CB15] 38 OXAH 663ZP_00058724.1, 659aa 0.00E+00 370/647 (57.19%) 435/647 (67.23%) Acyl-CoAdehydrogenases [Thermobifida fusca] AAB97825.1, 433aa 5.00E−93 203/446(45.52%) 251/446 (56.28%) acyl-CoA oxidase [Myxococcus xanthus]AAF14635.1, 694aa 5.00E−85 211/565 (37.35%) 292/565 (51.68%) 1 acyl-CoAoxidase [Petroselinum crispum] 39 ABCA 537 T14162, 574aa 9.00E−62189/509 (37%) 240/509 (47%) hABC transport protein - Mycobacteriumsmegmatis NP_624808.1 4.00E−60 184/540 (35%) 251/540 (46%) ABCtransporter [Streptomyces coelicolor A3(2)] NP_822745.1 8.00E−32 124/392(31%) 168/392 (42%) ABC transportert [Streptomyces avermitilis MA-4680]40 ABCA 596 T14180, 1122aa 1.00E−107 236/594 (39.73%) 300/594 (50.51%)exiT protein - Mycobacterium smegmatis AAC82548.1, 589aa 1.00E−107234/583 (40.14%) 295/583 (50.6%) unknown [Mycobacterium smegmatis]NP_624810.1, 601aa 3.00E−97 222/593 (37.44%) 283/593 (47.72%)ABC-transporter [Streptomyces coelicolor A3(2)] 41 UNIQ 507 NP_831570.1,676aa 8.00E−07  62/262 (23.66%) 116/262 (44.27%) methyltransferases[Bacillus cereus NP_655735.1, 676aa 2.00E−06  61/262 (23.28%) 116/262(44.27%) ubiE/COQ5 methyltransferase family [Bacillus anthracisNP_844290.1, 681aa 2.00E−06  61/262 (23.28%) 116/262 (44.27%)hypothetical protein [Bacillus anthracis str. Ames] 42 232 NP_830809.1,208aa 8.00E−08  46/210 (21.9%)  74/210 (35.24%) Transporter, LysE family[Bacillus cereus] NP_844737.1, 210aa 2.00E−07  46/210 (21.9%)  74/210(35.24%) homoserine/threonine efflux protein [Bacillus anthracisNP_655752.1, 208aa 1.00E−06  47/210 (22.38%)  75/210 (35.71%) LysE, LysEtype translocator [Bacillus anthracis 43 132 NP_827272.1, 127aa 4.00E−09 38/107 (35.51%)  52/107 (48.6%) hypothetical protein [Streptomycesavermitilis MA-4680] NP_246491.1, 112aa 5.90E−02  21/94 (22.34%)  44/94(46.81%) unknown [Pasteurella multocida]

The ORFs encoding proteins involved in the biosynthesis of compounds ofFormula II are assigned a putative function and grouped together infamilies based on sequence similarity to known proteins. To correlatestructure and function, the protein families are given a four-letterdesignation used throughout the description and figures as indicated inTable 15. The meaning of the four letter designations is as follows:AAKD designates an amino acid kinase; ABCA and ABCC designate ABCtransporters; ADSA designates an amide synthetase; ALDB designates analdolase function; CSMB designates a chorismate transaminase; DAHPdesignates a 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphatesynthase activity; DHBS designates a 2,3-dihydro-2,3-dihydroxybenzoatesynthase activity; DMDA designates a diphosphomevalonate decarboxylase;EFFT designates an efflux protein; HMGA designates a3-hydroxy-3-methylglutaryl-CoA reductase; HOXV designates amonooxygenase activity; HOYH designates a hydroxylase/decarboxylaseactivity; HYDK designates a hydrolase activity; IDSA designates anisopentenyl diphosphate synthase; IPPI designates an isopentenyldiphosphate isomerase; IPTN designates an isoprenyltransferase; KASHdesignates 3-hydroxy-3-methylglutaryl-CoA synthase; MVKA designates amevalonate kinase; MVPK designates a phosphomevalonate kinase; OXAHdesignates an acylCoA oxidase; OXDS designates an oxidoreductase; RECH,RECI, REGD, REGG and RREB designate regulators; SDRA designates adehydrogenase/ketoreductase, SPKG designates a sensory protein kinase;UNES, UNEZ, UNFA, UNFC, UNFD, UNFE, UNFJ and UNIQ designate proteins ofunknown function.

TABLE 15 FAMILY FUNCTION AAKD amino acid kinase; strong homology toprimary aspartate kinases, converting L- aspartate to4-phospho-L-aspartate ABCA ABC transporter ABCC ABC transporter ADSAadenylating amide synthetase ALDB aldolase; similarity tofructose-1,6-biphosphate aldolase that generates D- glyceraldehyde-3Ph,precursor of D-erythrose-4Ph involved in the shikimate pathway CSMBchorismate transaminase, similarity to anthranilate synthase DAHP DAHPsynthase, class II; involved in formation of aminoDAHP from PEP anderythrose-4-phosphate DHBS 2,3-dihydro-2,3-dihydroxybenzoate synthase(isochorismatase) DMDA diphosphomevalonate decarboxylase (mevalonatepyrophosphate decarboxylase) EFFT efflux protein HMGA HMG-CoA reductase;converts 3-hydroxy-3-methylglutaryl-CoA to mevalonate plus CoA inisoprenoid biosynthesis HOXV FAD monooxygenase; shows homology to avariety of monooxygenases including salicylate hydroxylases, zeaxanthinepoxidases HOYH hydroxylase/decarboxylase; FAD-dependent monooxygenaseHYDK hydrolase IDSA isoprenyl diphosphate synthase, catalyzes theaddition of 2 molecules of isopentenyl pyrophosphate to dimethylallylpyrophosphate to generate GGPP IPPI isopentenyl diphosphate isomerase,catalyzes the isomerization of IPP to produce dimethylallyl diphosphateIPTN isoprenyltransferase; catalyzes covalent N-terminal attachment ofisoprenyl units to amide groups of nitrogen-containing heterocycle ringsKASH HMG-CoA synthase; condenses acetyl-CoA with acetoacetyl-CoA to form3- hydroxy-3-methylglutaryl-CoA MVKA mevalonate kinase; convertsmevalonate to 5-phosphomevalonate in the mevalonate pathway ofisoprenoid biosynthesis MVKP phosphomevalonate kinase; converts5-phosphomevalonate to 5- diphosphomevalonate in the mevalonate pathwayof isoprenoid biosynyhesis OXAH acyl CoA oxidase OXDS oxidoreductaseRECH regulator RECI regulator; similarity to PadR transcriptionalregulators involved in repression of phenolic acid metabolism REGDtranscriptional regulator; relatively large regulators with anN-terminal ATP-binding domain containing Walker A and B motifs and aC-terminal LuxR type DNA-binding domain REGG regulator RREB responseregulator; similar to response regulators that are known to bind DNA andact as transcriptional activators SDRA dehydrogenase/ketoreductase,NAD-dependent SPKG sensory protein kinase, two component system UNESunknown function UNEZ unknown function UNFA unknown function UNFCunknown function UNFD unknown function UNFE putative membrane proteinUNFJ unknown function UNIQ unknown function

Biosynthesis of the compound of Formula II involves the action ofvarious enzymes that synthesize the three building blocks of thecompound, namely the farnesyl-diphosphate component (FIG. 13), the3-hydroxy-anthranilate-adenylate component (FIG. 14 a) and the2-amino-6-hydroxy-benzoquinone component (FIG. 14 b) that aresubsequently condensed to form the final compound (FIG. 15).

The farnesyl-diphosphate biosynthesis involves the concerted action ofseven enzymes (FIG. 13). ORF 10 (KASH) (SEQ ID NO: 20) encodes ahydroxymethylglutaryl-CoA synthase that catalyzes an aldol addition ofacetyl-CoA onto acetoacyl-CoA to yield 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA). This product is subsequently reduced through the action ofORF 9 (HMGA) (SEQ ID NO: 18) to form mevalonic acid (MVA). ORF 5 (MVKA)(SEQ ID NO: 10) phosphorylates mevalonate to 5′-phosphomevalonate usingATP as the phosphate donor. The next step in the farnesyl-diphosphatebiosynthesis is the phosphorylation reaction of the 5′-phosphomevalonateto 5′-pyrophosphomevalonate (DPMVA) that is catalyzed by ORF 7 (MVKP)(SEQ ID NO: 14). Subsequent decarboxylation of 5′-pyrophosphomevalonatecatalyzed by ORF 6 (DMDA) (SEQ ID NO: 12) yields isopentenyl diphosphate(IPP) which is then converted to dimethylallyldiphosphate (DMADP)through the action of ORF 8 (IPPI) (SEQ ID NO: 16) that has isomeraseenzymatic activity. The final step in the biosynthesis offarnesyl-diphosphate is the condensation of one molecule ofdimethylallyldiphosphate with two molecules of isopentenyl diphosphatecatalyzed by the isoprenyl diphosphate synthase ORF 4 (IDSA) (SEQ ID NO:8). The described pathway involved in synthesis of farnesyl-diphosphateis entirely consistent with related mevalonate pathways described inother actinomycete species (Takagi et al., J. Bacteriol. 182, 4153-4157,(2000)).

Biosynthesis of the 3-hydroxy-anthranilate component involves the use ofprecursors derived from the shikimate pathway (FIG. 14 a). Chorismicacid is transaminated through the action of ORF 19 (CSMB) (SEQ ID NO:38) to form aminodeoxyisochorismic acid. This enzyme resemblesanthranilate synthases and is likely to catalyze specifically thetransfer of the amino group using glutamine as the amino donor. The nextstep involves isochorismatase activity and is mediated by ORF 27 (DHBS)(SEQ ID NO: 54). This reaction consists in the removal of the pyruvateside chain from aminodeoxyisochorismic acid to form6-amino-5-hydroxy-cyclohexa-1,3-dienecarboxylic acid. This compound issubsequently oxidized through the action of ORF 26 (SDRA) (SEQ ID NO:52) yielding 3-hydroxy-anthranilic acid. ORF 24 (ADSA) (SEQ ID NO: 48)catalyzes the activation of 3-hydroxy-anthranilic acid throughadenylation generating the 3-hydroxy-anthranilate-adenylate component(FIG. 14 a).

Biosynthesis of the 2-amino-6-hydroxy-benzoquinone component of thecompound of Formula II, requires components derived from theaminoshikimate pathway. FIG. 14 b depicts the series of enzymaticreactions involved in the biosynthesis of this constituent. ORF 21(ALDB) (SEQ ID NO: 42) resembles aldolases involved in the generation ofprecursors of D-erythrose-4-phosphate which is part of theaminoshikimate pathway used for the generation of2-amino-6-hydroxy-[1,4]-benzoquinone. ORF 33 (DAHP) (SEQ ID NO: 67)catalyzes the initial step in the aminoshikimate pathway thatcorresponds to the formation of3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (amino DAHP)from phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E-4Ph).Subsequent reactions leading to 3-amino-5-hydroxy-benzoic acid arecatalyzed by enzymes provided by primary metabolism biosyntheticpathways present in Micromonospora sp. strain 046-ECO11. ORF 25 (HOXV)(SEQ ID NO: 50) hydroxylates 3-amino-5-hydroxy-benzoic acid at position2, generating 3-amino-2,5-dihydroxy-benzoic acid. This intermediate isfurther modified by ORF 32 (HOYH) (SEQ ID NO: 65) that catalyzes adecarboxylative oxidation reaction yielding 6-amino-benzene-1,2,4-triol.A final oxidation reaction is performed by ORF 16 (OXDS) (SEQ ID NO: 32)yielding 2-amino-6-hydroxy-[1,4]-benzoquinone (FIG. 14 b).

Assembly of the three components resulting in the compound of Formula IIis catalyzed by ORFs 24 and 11 (FIG. 15). ORF 24 (ADSA) (SEQ ID NO: 48)catalyzes the condensation of the adenylated 3-hydroxy-anthranilate withthe 2-amino-6-hydroxy-[1,4]-benzoquinone component. A spontaneouscondensation between the free amino group of the 3-hydroxy-anthranilateand one of the carbonyl groups present on the2-amino-6-hydroxy-[1,4]-benzoquinone component occurs yielding adibenzodiazepinone intermediate. This compound is further modifiedthrough transfer of the farnesyl group of the farnesyl-diphosphateintermediate onto the nitrogen of the amide of the dibenzodiazepinonecatalyzed by ORF 11 (IPTN) (SEQ ID NO: 22) and resulting in theformation of the compound of Formula II (FIG. 15).

Additional ORFs, namely ORF 2 (RECH) (SEQ ID NO: 4), ORF 3 (REGD) (SEQID NO: 6), ORF 12 (SPKG) (SEQ ID NO: 24), ORF 13 (RREB) (SEQ ID NO: 26),ORF 34 (REGG) (SEQ ID NO: 69) and ORF 36 (RECI) (SEQ ID NO: 74) areinvolved in the regulation of the biosynthetic locus encoding thecompound of Formula II. Other ORFs, namely ORF 1 (ABCC) (SEQ ID NO: 2),ORF 31 (EFFT) (SEQ ID NO: 62), ORFs 39 and 40 (ABCA) (SEQ ID NOS: 80 and82, respectively) and ORF 42 (SEQ ID NO: 86) are involved in transport.Other ORFs involved in the biosynthesis of the compound of Formula IIinclude ORF 20 (AAKD) (SEQ ID NO: 40), ORF 23 (HYDK) (SEQ ID NO: 46),ORF 38 (OXAH) (SEQ ID NO: 78) as well as ORFs 14, 15, 17, 18, 22, 29,30, 35, 37, 41 and 43 (SEQ ID NOS: 28, 30, 34, 34, 44, 58, 60, 71, 76,84 and 88, respectively) of unknown function.

TABLE 16 PREFERRED MEDIA COMPOSITION FOR PRODUCTION OF ECO-04601COMPONENT QB MA KH RM JA FA pH*⁵ 7.2 7.5 7 6.85 7.3 7.0 Glucose 12 10 1010 Sucrose 100 Lactose Cane molasses 15 Corn starch 30 Soluble starch 1025 Potato dextrin 20 40 Corn steep solid Corn steep 5 15 Dried yeast 2Yeast extract 5 Malt extract 35 Pharmamedia ™ 10 15 Glycerol NZ-Amine 510 Soybean 15 Soybean flour Meat extract Bacto-peptone MgSO₄•7H₂O 1MgCl₂•6H₂O CaCO₃ 4 1 2 2 NaCl 5 (NH₄)₂ SO₄ 2 K₂SO₄ 0.25 MnCl₂•4H₂OMgCl₂•6H₂O 10 FeCl₂•4H₂O ZnCl₂ Na₂HPO₄ 3 Thiamine Casamino acid 0.1Proflo oil 4 MOPS 21 Trace element 2 solution*³ ml/L Unless otherwiseindicated all the ingredients are in gm/L. *³Trace elements solutioncontains: ZnCl₂ 40 mg; FeCl₃ 6H₂O (200 mg); CuCl₂ 2H₂O (10 mg);MnCl₂•4H₂O; Na₂B₄O₇•10H₂O (10 mg); (NH₄)₆ MO₇O₂₄•4H₂O (10 mg) per litre.*⁵The pH is to adjusted as marked prior to the addition of CaCO₃.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1-4. (canceled)
 5. Cosmid 046KQ deposited under IDAC accession no.250203-07.
 6. A prokaryotic host cell comprising the cosmid of claim 5.7. The prokaryotic host cell of claim 6, wherein said host cell isselected from the group consisting of E. coli, Streptomyces lividans,Streptomyces griseofuscus, Streptomyces ambofuchsus, Streptomycesambofaciens, Actinomycetes, Bacillus, Corynebacteria andThermoactinomyces.
 8. An isolated DNA molecule having 95% sequenceidentity over the entire length of an open reading frame of the cosmidof claim
 5. 9-12. (canceled)